HAL Id: tel-03598649https://tel.archives-ouvertes.fr/tel-03598649

Submitted on 5 Mar 2022

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Functional interactions of nuclear RNase P inArabidopsis thaliana

Mathieu Bruggeman

To cite this version:Mathieu Bruggeman. Functional interactions of nuclear RNase P in Arabidopsis thaliana. VegetalBiology. Université de Strasbourg, 2020. English. �NNT : 2020STRAJ031�. �tel-03598649�

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G0!<%=!,*+2!,'.!*1!)%&!9%'+'9)&+-@')-*1!*6!)%&!6(19)-*1'/!-1)&+'9)-*1.!*6!1(9/&'+!OP'.&!

<!-1!84#0(9/-)()*%3#:(#$#>!L1!)%-.!;*4&/!7/'1)8!OP'.&!<!'9)-3-)0!-.!7&+6*+;&4!-1!)%&!1(9/&(.!

50!),*!+&4(14'1)!7+*)&-1.!9'//&4!<OYO<"!'14!#>!$%&!*3&+'//!'-;!*6!;0!,*+2!,'.!)*!4&6-1&!

)%&! 7+*)&-1! 7'+)1&+.! *6! Q)<OYO<"! '14! #! -1! *+4&+! )*! -4&1)-60! %*,! OP'.&! <! '9)-3-)0! -.!

-1)&:+')&4!,-)%!*)%&+!9&//(/'+!7+*9&..&.!'14!)*!4&)&+;-1&!-6!7/'1)!1(9/&'+!OP'.&!<!%'.!

*)%&+!.(5.)+')&.!5&.-4&.!7+&9(+.*+!)OPQ.>!

!

L1! *+4&+! )*! 7+&.&1)! )%&! 9(++&1)! .)')&! *6! 21*,/&4:&! *1! )%&! 5-*/*:-9'/! 6(19)-*1.! '14!

;&9%'1-.;.!+&/')&4!)*!;0!<%=!7+*e&9)8!L!,-//!6-+.)!7+&.&1)!%*,!)OPQ.!'+&!)+'1.9+-5&4!'14!

;')(+')&4>!$%&1!)%&-+!.)+(9)(+&!'14!6(19)-*1!,-//!5&!4&.9+-5&4!ILM>!P&B)8!L!,-//!7+&.&1)!)%&!

4-.)+-5()-*18!4-3&+.-)0!'14!6(19)-*1.!*6!)OPQJ/-2&!.)+(9)(+&.!ILLM>!S-19&!1(9/&'+!)OPQ.!'+&!

)+'1.9+-5&4!50!OPQ!7*/0;&+'.&!LLL!IOPQ!7*/!LLLM8!L!,-//!7+&.&1)!)%&!.7&9-6-9!6&')(+&.!*6!)%-.!

&1@0;&8!'.!,&//!'.!)%&!3'+-&)0!*6!OPQ.!)%')!)%-.!7*/0;&+'.&!)+'1.9+-5&.!ILLLM>!GQAU!-.!)%&!

;'-1!21*,!1&:')-3&!+&:(/')*+!*6!OPQ!7*/!LLL8!'14!*(+!,*+2!%'.!(19*3&+&4!'!7*..-5/&!7/'1)!

.7&9-6-9!+&:(/')-*1!*6!-).!'9)-3-)0!50!1(9/&'+!OP'.&!<>!H*1.&R(&1)/08!L!,-//!7+&.&1)!GQAU!

6(19)-*1!'14!;*4&!*6!'9)-*1!')!)%&!9&//(/'+!'14!;*/&9(/'+!/&3&/!ILKM>!$%&1!L!,-//!7+&.&1)!-1!

'!/'.)!7'+)!*6!)%&!-1)+*4(9)-*18!),*!4-66&+&1)!.)&7.!*6!)OPQ!;')(+')-*1!)%')!'+&!.7&9-6-9'//0!

+&/')&4!)*!;0!<%=>!A-+.)8!L!,-//!7+&.&1)!-1!4&)'-/.!)%&!4-3&+.-)0!*6!OP'.&!<!&1@0;&.!)%')!

remove the 5’ leader sequence of precursor tRNA!IKM>!!$%&18!L!,-//!7+&.&1)!;*+&!5+-&6/0!

)%&! 9%&;-9'/! ;*4-6-9')-*1! *6! )OPQ.! 1(9/&*)-4&.! '14! ;*+&! .7&9-6-9'//08! )%&! +&'9)-*1!

9')'/0.&4!50!'1!&1@0;&!9'//&4!$+;U8!)%')!,'.!.%*,1!-1!)%-.!,*+28!)*!5&!'!7+*)&-1!7'+)1&+!

*6!Q)<OY<"d#!IKLM>!

! U_!

!

BO ,8@!-X./2%*($(-2-`.-,0L",L0(.3$).WL$",2%$-.

!

First characterized in 1958 as “soluble ribonucleic acid intermediates in protein

synthesis” (Hoagland et al., 1958), tRNAs were the first nonJ9*4-1:! OPQ.! I19OPQ.M!

4-.9*3&+&4>! )OPQ.! %'3&! '! 3&+0! .7&9-6-9! .)+(9)(+&! I'M8! )%')! -.! 9/*.&/0! +&/')&4! )*! )%&-+!

6(19)-*1>!$%&!;'-1!+*/&!*6!)OPQ.!-.!)*!4&/-3&+!';-1*!'9-4.8!'.!.7&9-6-&4!50!;&..&1:&+!OPQ!

I;OPQM! 9*4*1.8! )*! +-5*.*;&.>! ?*,&3&+8! )OPQ.! %'3&! '44-)-*1'/! 6(19)-*1.! )%')! '+&!

7+*:+&..-3&/0!(19*3&+&4!I5M>!A-1'//08!)%&!4-66&+&1)!7*.)J)+'1.9+-7)-*1'/!.)&7.!-13*/3&4!-1!

)OPQ!5-*:&1&.-.!'14!)(+1*3&+!,-//!5&!7+&.&1)&4!I9M>!

!

#? %@;8*)%4"<%"4.*!tRNAs have a conserved secondary structure often referred to as a “cloverleaf’. Their 3D

'+9%-)&9)(+&! -.! '1! DJ.%'7&4! 6*/4! )%')! -.! '/.*! 9*1.&+3&4! IA-:(+&! UM>! ?*,&3&+8! )%-.! #=!

.)+(9)(+&!-.!+')%&+!401';-9!'14!1*)!6-B&48!'.!'1'/0.&.!*6!9+0.)'/!.)+(9)(+&!'14!9+0*JZG!

+&9*1.)+(9)-*1! *6! )OPQ.! .)+(9)(+&.! -1! 9*;7/&B! ,-)%! 4-66&+&1)! 7'+)1&+.! I7+*9&..-1:!

&1@0;&.8! +-5*.*;&.8! )OPQ! .01)%&)'.&.M! .%*,! )%')! )OPQ.! 9'1! 4-.7/'0! '! /'+:&! +'1:&! *6!

.)+(9)(+'/!'++'1:&;&1).!)*!7&+6*+;!)%&-+!6(19)-*1.!I]&.)%*6!'14!Q(66-1:&+8!"_U"M>!

)OPQ.!/&1:)%!3'+0!6+*;!'+*(14!g_!)*!U__!1(9/&*)-4&.>!Q.!1*)&4!50!]&.)%*6!'14!Q(66-1:&+8!

I"_U"M8!)%&0!'+&!&3*/()-*1'+0!9*1.)+'-1&4!50!),*!*77*.-)&!6*+9&.>!$%&0!;(.)!%'3&!(1-R(&!

6&')(+&.!-1!*+4&+!6*+!)%&-+!9*:1')&!)OPQ!.01)%&)'.&!)*!';-1*'90/')&!)%&!9*++&9)!)OPQ!,-)%!

)%&!.7&9-6-&4!';-1*!'9-4!9*++&.7*14-1:!)*!-).!'1)-9*4*1>!Q14!*1!)%&!*)%&+!%'148!)%&-+!#=!

.)+(9)(+&!-.!9*1.)+'-1&4!50!)%&!1&9&..-)0!)*!6-)!-1!)%&!+-5*.*;&.!5-14-1:!.-)&.8!-1!*+4&+!6*+!

9*++&9)! 7+*)&-1! .01)%&.-.! )*! *99(+>! Q.! .(9%8! )OPQ! .)+(9)(+&! %'+5*(+.! '! 9*;;*1!

'+9%-)&9)(+&!-1.-4&!,%-9%!;*/&9(/'+!4-3&+.-)0!&B-.).!)*!&1.(+&!)%&!(1-9-)0!*6!&'9%!)OPQ>!!

$%&!9/*3&+/&'6!.&9*14'+0!.)+(9)(+&!-.!;'4&!(7!*6!6*(+!%&/-9&.8!)%+&&!/**7.!'14!'!3'+-'5/&!

4*;'-1>!A-+.)8!)%&!'99&7)*+!.)&;!I'/.*!9'//&4!'99&7)*+!%&/-BM!)%')!9'++-&.!)%&!';-1*!'9-4!')!

its 3’ end is usually made of seven base pairs. The first four base pairs of the acceptor!

! UU!

..F2*L0(.TN!45.3$).65.-"R(+3,2".0(P0(-($,3,2%$.%W.3.,8@!>!'M!H/*3&+/&'6!4-':+';!*6!)%&!"=!6*/4-1:!7'))&+1!*6! '! )OPQ>!P(;5&+.! -14-9')&! )%&! 1(9/&*)-4&! 5'.&8! '/;*.)! (1-3&+.'//0! 6*(14! ')! )%-.! 7*.-)-*1! -1! '//! )OPQ!.&R(&19&.! 21*,1! )*! 4')&>! Q! a! '4&1*.-1&j! \! a! :('1*.-1&j! H! a! 90)-4-1&j! E! a! (+-4-1&j! O! a! '4&1*.-1&! *+!:('1*.-1&j!V!a!90)-4-1&!*+!(+-4-1&j!$!a!+-5*)%0;-4-1&j!'14!Y a!7.&(4*(+-4-1&>!5M!A*/4-1:!7'))&+18!-1!#=8!*6!0&'.)!7%&10/'/'1-1&!)OPQ>!$%&!9*-/&4!)(5&!-14-9')&.!)%&!.(:'+J7%*.7%')&!5'925*1&!*6!)%&!;*/&9(/&>!$%&!9+*..!+(1:.!+&7+&.&1)!)%&!1(9/&*)-4&!5'.&!7'-+.!,%-/&!)%&!.%*+)!+(1:.!4&7-9)!5'.&.!1*)!-13*/3&4!-1!5'.&J5'.&!%04+*:&1!5*14-1:>!$%&!9*/*(+.!-1!)%&!#=!4-':+';!+&6&+!)*!)%&!"=!.)+(9)(+&>!=-':+';.!'+&!+&7+*4(9&4!'14!'4'7)&4!6+*;!O-9%!'14!`-;!IUbgfM!'14!\*/4;'1!I"__fM>!! !

! U"!

%&/-B!'+&!-;7*+)'1)!6*+!)OPQ!.01)%&)'.&!+&9*:1-)-*1!I\-&:F!&)!'/>!"_U"M!'.!1*)&4!'5*3&>!

Position 73, located just before the CCA is called the ‘discriminator’, due to its role in the

9*++&9)! ';-1*'90/')-*1! *6! )OPQ.>! $%&1! *1! the 5’ side of the cloverleaf,! )%&! =!

I4-%04+*(+-4-1&M!'+;!-.!1';&4!'6)&+!)%-.!;*4-6-&4!5'.&!)%')!*99(+.!-1!-).!/**7>!$%&!6-+.)!

)%+&&!5'.&!7'-+.!*6!)%&!=!%&/-B!'/.*!9*1.)-)()&!-4&1)-)0!&/&;&1).!6*+!';-1*'90/!.01)%&)'.&.>!

$%&18!)%&!'1)-9*4*1!'+;!9*1)'-1.!-1!-).!/**7!)%&!'1)-9*4*1!)+-7/&)!)%')!,-//!7'-+!,-)%!)%&!

9*;7/&;&1)'+0!9*4*1!*6!)%&!;OPQ!4(+-1:!7+*)&-1!.01)%&.-.!-1!)%&!+-5*.*;&>!$%&!-4&1)-)0!

*6!)%&!+&.-4(&.!*6!)%&!'1)-9*4*1!/**7!'+&!'/.*!-;7*+)'1)!6*+!9*++&9)!';-1*'90/')-*1>!$%&!

'1)-9*4*1!'+;!-.!6*//*,&4!50!)%&!3'+-'5/&!+&:-*1!)%')!9*1)'-1.!5&),&&1!T!'14!"U!+&.-4(&.>!

$%-.!3'+-'5/&!/**7!-.!)%&!;'e*+!.*(+9&!*6!3'+-'5-/-)0!-1!)OPQ!.-@&.8!%&19&!-).!1';&>!$%&!/'.)!

9*1.&+3&4!4*;'-1!4&6-1-1:!)OPQ.!-.!)%&!$J'+;8!1';&4!'6)&+!'!)%0;-4-1&!7+&.&1)!-1!)%&!

9*1.&+3&4!$YC motif of its loop. Finally, the 3’end the mature tRNA is always termi1')&4!

50!)%&!HHQ!.&R(&19&!,%&+&!)%&!';-1*!'9-4!-.!'))'9%&4>!

)OPQ!.&R(&19&.!'/-:1;&1)!.%*,!)%')!-1!)%&!"=!.)+(9)(+&8!.*;&!7*.-)-*1.!1&'+/0!'/,'0.!

consist of the same type of base (called ‘invariants’). Some other positions are conserved

'.!&-)%&+!7(+-1&.!*+!70+-;-4-1es (called ‘semiJinvariants’) (F-:(+&!"M>!L13'+-'1)!'14!.&;-J

-13'+-'1)!7*.-)-*1.!+&7+&.&1)!(7!)*!"_!1(9/&*)-4&.!-1!)OPQ.>!$%&!#=!.)+(9)(+&!*6!)OPQ.!

.)+*1:/0!+&/-&.!*1!)%&!6*(+!%&/-9&.!4&.9+-5&4!'5*3&>!$%&!'99&7)*+!%&/-B!'14!)%&!)%0;-4-1&!

'+;! .)'92! )*:&)%&+! )*! 6*+;! )%&! '99&7)*+!4*;'-1!,%-/&! )%&! 4-%04+*(+-4-1&! '+;! .)'92.!

)*:&)%&+!,-)%!)%&!'1)-9*4*1!'+;!)*!;'2&!)%&!'1)-9*4*1!4*;'-1>!$%-.!.)+(9)(+&!-.!*3&+'//!

.%'7&4!/-2&!'1!D8!'.!)%&!),*!4*;'-1.!6*+;.!'1!'1:/&!*6!'+*(14!b_k>!$%-.!#=!.)+(9)(+&!-.!

;'-1)'-1&4!50!/**7J/**7!)&+)-'+0!9*1)'9).!I5&),&&1!)%&!$!'14!=!/**7.M!'14!50!)&+)-'+0!

9*1)'9).! I-13*/3-1:! 1*1J]').*1JH+-92! 7'-+-1:.! '14! )%&! 6*+;')-*1! *6! 5'.&! )+-7/&).M!

5&),&&1!)%&!.-1:/&!.)+'14&4!e(19)-*1.!/-12-1:!)%&!%&/-9'/!4*;'-1.!'14!)%&!4&&7!:+**3&!*6!

)%&!=!%&/-B>! )OPQ!.)+(9)(+&.!'+&!'/.*! +-:-4-6-&4!50!;':1&.-(;!9')-*1.! IQ(66-1:&+!&)! '/>8!

"_UUM>!L14&&48!-)!,'.!.%*,1!)%')!-1!)%&!'5.&19&!*6!G:"c8!.*;&!)OPQ.!9*(/4!1*)!5&!';-1*J

'90/')&4!IG'4*+&!&)!'/>8!UbbbM>!$%&!+*/&!*6!;':1&.-(;!9')-*1.! -.!'/.*! -;7*+)'1)! 6*+8!'.!

.)')&4!'5*3&8!)%&!401';-9!.)+(9)(+'/!9%'1:&.!)%')!)OPQ.!9'1!'4'7)!,%&1!-1)&+'9)-1:!,-)%!

)%&-+!4-66&+&1)!7+*)&-1!7'+)1&+.!I]&.)%*6!'14!Q(66-1:&+8!"_U"M>!

Q//!)%&.&!&/&;&1).!7*-1)!*()!)%&!-;7*+)'19&!*6!)OPQ.!.)+(9)(+&>!L14&&48!-6!6*+!;OPQ!)%&!

.&R(&19&!-.!9*1.-4&+&4!'.!)%&!4&)&+;-1'1)!*6!-).!5-*/*:-9'/!6(19)-*18!6*+!)OPQ!-)!-.!)%&!!

! U#!

!

!F2*L0(.4N!@%+($"'3,L0(.3$)./3-(.)2-,02/L,2%$-.2$.('%$*3,%0.,8@!>!I'M!P(;5&+.!-14-9')&!)%&!'99&7)&4!1*;&19/')(+&!*6!)OPQ!;*/&9(/&.!'.!4&6-1&4!-1!S7+-1@/!&)!'/>8!Ubbf>!S)+'-:%)!5/'92!/-1&.!-14-9')&!.&9*14'+0!5'.&!7'-+-1:!,%-/&!5+*2&1!/-1&.!-14-9')&.!(1(.('/!5'.&!7'-+-1:>!I5M!=-.)+-5()-*1!*6!)%&!6*(+!9*;;*1!5'.&.!')!&'9%!7*.-)-*1!'.!9'/9(/')&4!6+*;!b#"!)OPQ!.&R(&19&.!50!Q(66-1:&+!'14!]&.)%*68!Ubbf>!L1!%&/-9&.8!)%&!9*/*(+!9*4&!6*+!7'-+&4!+&.-4(&.!'+&!'++'1:&4!.*!'.!)*!6*//*,!]').*1JCrick pairings. The 5’ strand has a thin outer circle while the 3’ strand has a thick outer circle (fr*;!]&.)%*6!'14!Q(66-1:&+8!"_U"M>!! !

! UT!

.)+(9)(+&!I->&!)%&!.7')-'/!6*/4!*6!)%&!OPQ!)*!6*+;!'!)&+)-'+0!.)+(9)(+&M!)%')!-.!)%&!5'.-.!*6!-).!

5-*/*:-9'/!'9)-3-)0!I]&.)%*6!'14!Q(66-1:&+8!"_U"M>!

!

0? %@;8)*0(/:/A(<#:*B"$<%(/$)*!

)OPQ.! 9'1*1-9'/! 6(19)-*1! -.! )*! 4&/-3&+! ';-1*! '9-4.8! '.! .7&9-6-&4! 50! ;&..&1:&+! OPQ!

I;OPQM!9*4*1.!)*!)%&!7+*)&-1!.01)%&.-.!;'9%-1&+-&.j!-1!)%&!90)*7/'.;!I6*+!<+*2'+0*)&.!

'14!1(9/&'+!&(2'+0*)-9!;OPQM!'14!-1!*+:'1&//&.!I-1!Z(2'+0*)&.!*1/0M>!$*!7&+6*+;!)%-.!

.)&78!)OPQ.!1&&4!)*!5&!I9*++&9)/0M!';-1*J'90/')&4>!L14&&48!)OPQ.!9'1!5&!.(54-3-4&4!-1)*!

families of isoacceptors. Each defined by the corresponding (usually called “cognate”)

';-1*! '9-4! -)! 9'++-&.>! Q;-1*'90/')-*1! -.! 7&+6*+;&4!50! '! .&)! *6! .7&9-6-9! 7+*)&-1.! 9'//&4!

';-1*'90/J)OPQ!.01)%&)'.&>!C'.&4!*1!.&R(&19&!'1'/0.-.!'14!'6)&+,'+4.!*1!.(5.&R(&1)!

.)+(9)(+&!-13&.)-:')-*1.!';-1*'90/J)OPQ!.01)%&)'.&.!I''OSM!'+&!4-3-4&4!-1!),*!4-.)-19)!

9/'..&.!I9/'..!L!'14!LLM8!5'.&4!*1!)%&-+!4-66&+&19&.!')!5*)%!)%&!.)+(9)(+'/!'14!.&R(&19&!/&3&/!

I`'-.&+!&)!'/>8!"_"_M>!D-12-1:!*6!)OPQ!;*/&9(/&.!,-)%!)%&-+!9*:1')&!';-1*!'9-4!-.!*1&!*6!

)%&!;*.)!9+(9-'/!.)&7!*6!7+*)&-1!.01)%&.-.8!'.!*19&!)%&!';-1*!'9-4!-.!9%'+:&4!)*!)%&!)OPQ8!

-)!,-//!5&!-19*+7*+')&4!-1!)%&!&/*1:')-1:!7&7)-4-9!9%'-1!-1!)%&!+-5*.*;&8!,%&)%&+!*+!1*)!-)!

-.!)%&!9*++&9)!*1&!I\-&:F!'14!Z+-'1-8!"_U"M>!Q;-1*'90/J)OPQ!.01)%&)'.&!+&9*:1-@&!)%&-+!

cognate tRNA by specific features defining “tRNA identity”. For some tRNAs, the major

-4&1)-)0!&/&;&1)!-.!)%&!'1)-9*4*1!I\*/4;'18!"__fM>!?*,&3&+8!)%&!;'-1!6&')(+&.!9'1!'/.*!

5&! -1! )%&!'99&7)*+!.)&;!,-)%! /-))/&! )*!1*!.7&9-6-9-)0!9*16&++&4!50!)%&!'1)-9*4*1>!S)-//8! -1!

;*.)!9'.&.8!)%&!;'e*+!.7&9-6-9!6&')(+&!.&&;.!)*!5&!)%&!'1)-9*4*1!,-)%!'44-)-*1'/!-4&1)-)0!

&/&;&1).!+&.-4-1:!-1!)%&!'99&7)*+!.)&;8!'14d*+!)%&!=J/**7!I\*/4;'18!"__f8!]&.)%*6!'14!

Q(66-1:&+8!"_U"M!IA-:(+&!#M>!H%'+:-1:!*6!)%&!9*:1')&!';-1*!'9-4!)*!)%&!)OPQ!-.!'!),*J.)&7!

7+*9&..>!A-+.)!)%&!';-1*!'9-4!-.!'9)-3')&4!)*!'1!';-1*'90/!'4&10/')&!I,-)%!'!%-:%!&1&+:0!

5*14M>!$%&1!)%-.!'9)-3')&4!;*/&9(/&!-.!5*(14!)*!)%&!';-1*'90/!)OPQ!.01)%&)'.&!'14!)%&!

';-1*!'9-4! -.! )+'1.6&++&4!)*!)%&!)OPQ!I)%&!%-:%!&1&+:0!5*14!-.!7+&.&+3&48!'.! -)!,-//!5&!

1&9&..'+0! 6*+!7+*)&-1! .01)%&.-.M! IA-:(+&!TM>!=(&! )*!)%&!&B-.)&19&!*6!7+**6+&'4-1:! I'/.*!

called “editing”) mechanisms, that allow amino'90/!)OPQ!.01)%&)'.&!)*!+&e&9)!-19*++&9)!

';-1*'90/! '4&10/')&.8! )%&! ';-1*'90/')-*1! 7+*9&..! -.! %-:%/0! '99(+')&>! L14&&48! )%&!

;-.9%'+:-1:!6+&R(&190!%'.!5&&1!&.)-;')&4!')!/&..!)%'1!U_J[!I\*/4;'18!"__fM>!

! U^!

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tRNA takes place in the peptidyl (‘P’ site) of the 80S ribosome, and allows the binding of

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eIF4F (a complex formed of eIF4E, eIF4G and eIF4A) at its 5’ mg!IgJ;&)%0/:('1*.-1&M!9'7!

and of the PABP (poly(A) binding protein) on the 3’ poly(A) tail. After scanning of the PIC

on the mRNA, a ‘closed’ complex will!6*+;!,%&1!5'.&!7'-+-1:!-.!&.)'5/-.%&4!5&),&&1!)%&!

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protein synthesis (called “elongation”) requires only a minimal set of proteins (called

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(called the “peptidyl transferase” reaction or ‘peptide bond formation’)!9*;&.!6+*;!)%&!

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translocate to the P site while the one in the P site will translocate to the exit (‘E’) site.

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)%&!HHQ!)+-7/&)!'14!1(;&+*(.!9%&;-9'/!;*4-6-9')-*1.!*6!1(9/&*.-4&.>! L1!0&'.)!(7!)*!"^!

5'.&!;*4-6-9')-*1.!%'3&!5&&1!9%'+'9)&+-@&4!I?*77&+8!"_U#M!

Removal of the 5’ leader sequence is catalysed by RNase P in all domains *6!/-6&>!L1!0&'.)!

OP'.&! <! %'.! -1)&+&.)-1:! 6&')(+&.>! S-;-/'+! )*! )%&! '19&.)+'/! 5'9)&+-'/! +-5*1(9/&*7+*)&-1!

OP'.&!<8!5*)%!)%&!1(9/&'+!'14!;-)*9%*14+-'/!&1@0;&.!'+&!9*;7*.&4!*6!OPQ!'14!7+*)&-1.>!

]%-/&! 1(9/&'+! OP'.&! <! 9*1.-.).! *6! '! .-1:/&! 9')'/0)-9! OPQ! '14! 1-1&! 7+*)&-1.! I?*77&+8!

"_U#M8!;-)*9%*14+-'/!OP'.&!<!-.!9*;7*.&4!*6!'!4-66&+&1)!OPQ!'14!*1&!7+*)&-1!.(5(1-)!%'.!

5&&1!-4&1)-6-&4!)*!4')&!I='*(48!"_U"M>!L1)&+&.)-1:/08!;*.)!*6!)%&!7+*)&-1.!*6!0&'.)!I'14!

%(;'1M!OP'.&!<!'+&!.%'+&4!,-)%!OP'.&!GO<8!'1!&1@0;&!-13*/3&4!-1!7+&J+OPQ!7+*9&..-1:!

IX-'*!&)!'/>8!"__"j!m'++*(.!'14!\*7'/'18!"_U_M>!S-19&!OP'.&!<!-.!9&1)+'/!)*!;0!<%=!7+*e&9)8!

L!,-//!7+&.&1)!-).!4-3&+.-)08!->&>!7/'1)!OP'.&!<8!-1!;*+&!4&)'-/.!-1!'!.&7'+')&!.&9)-*1!IKM>!

The 3’ trailer sequence of preJ)OPQ.!-.!+&;*3&4!-1!0&'.)!50!5*)%!&B*1(9/&'.&.!ID?<U!'14!

OZXUM! '14! '1! &14*1(9/&'.&! I$OWUM>! $%&! ),*! &B*1(9/&'.&.! ,&+&! .%*,1! )*! 5&! 1*1J

&..&1)-'/!I?*77&+8!"_U#M8!,%&+&'.!$OWU!-.!&19*4&4!50!'1!&..&1)-'/!:&1&!I$'2'2(!&)!'/>8!

"__#M>!L1!7/'1).8!)%&!+&;*3'/!*6!)%e 3’ trailer is c')'/0.&4!50!)%&!&14*1(9/&'.&!OP'.&!W>!

OP'.&!W!&B-.).!-1!),*!6*+;.>!S%*+)!3&+.-*1.!*6!"__!)*!T__!';-1*!'9-4.!9*J&B-.)!,-)%!/*1:!

3&+.-*1.!*6!g__!)*!b__!''!IH'1-1*!&)!'/>8!"__bM>!Y1/0!)%&!.%*+)!6*+;!*6!OP'.&!W!-.!6*(14!-1!

<+*2'+0*)&.!,%&+&'.!Z(2'+0*)&.!7*..&..!)%&!/*1:!6*+;!'14!.*;&)-;&!5*)%!'.!6*(14!-1!

7/'1).>!OP'.&!W!5&/*1:.!)*!)%&!.(7&+6';-/0!*6!;&)'//*JβJ/'9)';'.&.!IGCD.M8!9%'+'9)&+-@&4!

50!)%&ir αJβdβJα 6*/4>!G&)'//*JβJ/'9)';'.&.!9*1)'-1!6-3&!%-:%/0!9*1.&+3&4!;*)-6.!)%')!'+&!

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&)!'/>8!"__b8!.%*,&4!)%')!84#0(9/)-)()*:&1*;&!&19*4&.!)%&!T!.%*+)!'14!/*1:!3&+.-*1.!*6!

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;-)*9%*14+-'!'14!)%&!1(9/&(.8!'14!D"!*1/0!-1!;-)*9%*14+-'>!

! ""!

It is worth noting that RNase P’s action usually precedes RNase! W>!?*,&3&+8! )%&+&!'+&!

.*;&!21*,1!&B9&7)-*1.8!'.!4&.9+-5&4!&>:>!50!O’Connor and P&&5/&.!IUbbUM>!

A 3’ terminal CCA sequence is required in all tRNAs for aminoacylation. In most cases, this

HHQ!.&R(&19&!-.!'44&4!7*.)J)+'1.9+-7)-*1'//0!50!'!)OPQ!1(9/&*)-40/!)+'1.6&+'.&>!L)!9'1!5&!

1*)&4! )%')! -1! .*;&!5'9)&+-'8! ->&>!E,* </:(8! )%&! HHQ! .&R(&19&! -.! &19*4&4!,-)%-1! )%&! )OPQ!

:&1&.8!%*,&3&+8!'!)OPQ!1(9/&*)-40/!)+'1.6&+'.&!.)-//!&B-.).!'14!,'.!.%*,1!)*!5&!-13*/3&4!

in tRNA 3’end repair (Zhu and Deutscher, 1987). In ',* <.4.7()(#.C* )OPQ! 1(9/&*)-40/!

)+'1.6&+'.&!-.!&19*4&4!50!'!.-1:/&!:&1&!IHHQU8!Q&5-!&)!'/>!Ubb_M>!HHQU!&19*4&.!#!-.*6*+;.!

:&1&+')&4!50!'/)&+1')-3&!)+'1.9+-7)-*1'/!'14!)+'1./')-*1'/!.)'+)!.-)&.8!'14!/*9')&4!-1!)%&!

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)OPQ!1(9/&*)-40/)+'1.6&+'.&!,'.!'/.*!-4&1)-6-&4!IQ)U:""[[_M>!$%-.!7+*)&-1!/*9'/-@&.!)*!)%&!

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.)&7!*6!.7/-9-1:8!9')'/0.&4!50!)%&!"’ 7%*.7%*)+'1.6&+'.&8!&19*4&4!50!$<$U!IS7-1&//-!&)!'/>8!

UbbgM>! $%-.! /-:')-*1! ;&9%'1-.;! -.! 9*1.&+3&4! -1! 7/'1).! I\&:&1%&-;&+! &)! '/>8! Ubf#j!

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4-66&+&1)! )-;&! 4(+-1:! )%&! 7*.)J)+'1.9+-7)-*1'/! ;')(+')-*1! *6! )OPQ.8! '! ;(/)-)(4&! *6!

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“tRNAJlike structures” or TLS were first discovered in 1970 in plant viruses. Pinck et al.,

'14!V*)!&)!'/>8!IUbg_M!4-.9*3&+&4!)%')!)he 3’end of the Turnip yellow mosaic virus (TYMV)

OPQ!9'1!5&!';-1*'90/)&4!,-)%!3'/-1&>!S-19&!)%')!4')&8!$DS!%'3&!5&&1!4&6-1&4!'.!&-)%&+!'1!

OPQ!;*/&9(/&!)%')!9'1!+&'9)!&66-9-&1)/0!,-)%!*1&!*+!;*+&!)OPQJ.7&9-6-9!&1@0;&!*+!'.!'1!

OPQ!)%')!%'.!'!.&R(&19&!.-;-/'+!)*!)%&!*1&!*6!'!)OPQ8!;&'1-1:!)%')!-).!.&9*14'+0!'14!d!*+!

)&+)-'+0!.)+(9)(+&!9/*.&/0!+&.&;5/&.!)*!)%&!9'1*1-9'/!9/*3&+J/&'6!.&9*14'+0!.)+(9)(+&!*+!DJ

.%'7&4!)&+)-'+0!.)+(9)(+&!*6!'!)OPQ!IG'1.!&)!'/>8!UbbUM>!

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investigated today. More specifically, TLS are found at the 3’ end of the genome of some

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&B';7/&M8!*)%&+.!%'3&! .)+*1:/0!&3*/3&4!'14!1*,!%'3&!*1/0!9*1.&+3&4!“vestigial” tRNA

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telomerase to maintain intact 3’ CCA termini. Finally, they can also be involved in the

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;';;'/-'1!/*1:!1*1Jcoding RNAs MALAT1 and MENβ ($*7(7/>!

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in different human cancers (5’ end of the preJGQDQ$U!)+'1.9+-7).M!'14!'![U!1(9/&*)-4&.!

)OPQ!/-2&!.)+(9)(+&!5+*'4/0!&B7+&..&4!-1!%(;'1!)-..(&.>!

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.-1:/&!OPQ!7*/0;&+'.&!LL!)+'1.9+-7)!')!)%&!G&1!ε/β locus in human (Sunwoo et al. 2009).

$%-.!.-1:/&!7+&Jtranscript contains a 3’ polyJQ!+-9%!+&:-*1!/*9')&4!(7.)+&';!*6!'!)OPQJ/-2&!

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Menβ transcript containing a poly(A) rich 3’ end in addition to the Men ε that contains the

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`1*924*,1!*6!)%&!G&1!ε/β transcripts led to the disruption of paraspeckles, highlighting

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In plant mitochondria, numerous TLS have been described, i.e. in the 5’ or 3’ untranslated

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obtain the mature 3’ end of this mRNA (Gobert et al.8!"_U_8!\();'11!&)!'/>8!"_U"M>!

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$%-.!9*+&!&19/*.&.!'!9&1)+'/!9/&6)!,%&+&!)%&!=PQ!,-//!5&!/*9')&4!4(+-1:!)+'1.9+-7)-*1!'.!

,&//!'.!),*!9%'11&/.8!*1&!6*+!)%&!OPQ!7+*4(9)!'14!*1&!6*+!)%&!P$<.!.(5.)+')&.>!$%-.!9*+&!

-.! 9*1.-4&+&48! 50! .)+(9)(+'/! 5-*/*gists, as having a “crabJclaw” shape! IA-:(+&! bM>! H*+&!

elements also consist of two “pincers” called “clamp” and “jaw”. Their function is to

.)'5-/-@&!)%&!=PQ!')!)%&!4*,1.)+&';!&14!'14!)*!'//*,!9/*.-1:!'14!*7&1-1:!*6!)%&!9/&6)>!

=(+-1:! )+'1.9+-7)-*18! )%&! OPQ! 7*/0;&+'.&! ,-//! ;'-1)'-1! '! .*Jcalled “)+'1.9+-7)-*1!

5(55/&” with separated DNA strands. This will facilitate nucleotides addition, allow the

&1@0;&!)*!)+'1./*9')&!'/*1:!)%&!)&;7/')&!'14!.)'5-/-@&!)%&!%05+-4!5&),&&1!)%&!1'.9&1)!

OPQ! )+'1.9+-7)! '14! )%&! =PQ! )&;7/')&! I`%'))&+! &)! '/>8! "_UgM>! L1! '44-)-*1! )*! )%-.! 9*+&8!

eukaryotic RNA polymerases share two additional subunits that will form the “stalk”. And

'44-)-*1'/!7&+-7%&+-9!.(5(1-).!&B-.)!-1!7*/!L!'14!7*/!LLL!I2%'))&+!&)!'/>8!"_UgM>!!!

! #"!

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! ##!

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4&)'-/.M>!

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!

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that the vast majority of RNA pol. III transcripts have a uniform 3’end composed of a

.)+&)9%!of Us (Arimbasseri, 2018). The La proteins will bind to this 3’end, protecting it

6+*;!&B*1(9/&'.&!4&:+'4')-*1>!A*+!)OPQ.!)%-.!.)&7!,-//!4-+&9)!)%&!5&:-11-1:!*6!)%&-+!7*.)J

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2007) by protecting the 3’end against exonuclease.!and to direct it towards the 5’J6-+.)!

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matured at their 3’ end by exonuclease.!before removal of the 5’ leader (Yoo et al.8!UbbgM>!

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complementation assays and was able to bind to the 3’ poly(U) end of RNA pol III

)+'1.9+-7).!($*7(7/>!$%-.!7+*)&-1!,'.!1';&4!Q)D'U!IQ)T:#"g"_M!'14!,'.!.%*,1!)*!5&!'1!

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!

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+&R(-+&;&1)! 6*+! ),*! 7+*;*)&+! .&R(&19&.! ,-)%-1! )%&! )+'1.9+-5&4! .&R(&19&! I->&>!

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7*+)-*1! *6! )%&! )OPQ! :&1&! )%')! &19*4&.! )%&! =! '+;! I1(9/&*)-4&.! f–UbM8! ,%-/&! C*B! C!

generally corresponds to the portion of the tRNA gene that encodes the TΨC arm

I1(9/&*)-4&.!^"–62). Flanking sequences, primarily on the 5’ side and m*.)! 6+&R(&1)/0!

-19/(4-1:! '! $Q$Q! &/&;&1)8! 9'1! '/.*! :*3&+1! )%&! &66-9-&190! *6! )%&! )+'1.9+-7)-*1! 7+*9&..!

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)%&!JU!)*!JU_!+&:-*1!-.!7'+)-9(/'+/0!+-9%!-1!Q!'14!H!+&.-4(&.!'14!HQQ!)+-7/&).!,&+&!.%*,1!

)*!5&!)+'1.9+-7)-*1!-1-)-')-*1!.-)&.!IG-9%'(4!&)!'/>8!"_UUM>!

!

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0&'.)!'14!%(;'1!GQAU>!A(+)%&+;*+&8!)%&0!-13&.)-:')&4!)%&!&66&9)!*6!'(B-1!'.!-)!7+*;*)&.!

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%3#:(#$#>! $%&!;'-1! 9*19/(.-*1! -.! )%')! Q)G'6U! -.! +&:(/')&48! .-;-/'+! )*! *)%&+! .7&9-&.8! 50!

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! Tg!

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!

OP'.&!<8! )%&!&1@0;&!)%')!7&+6*+;.!the 5’ maturation of tRNA precursors and of RNAs

9*1)'-1-1:!)OPQJ/-2&!.)+(9)(+&.!%'.!5&&1!')!)%&!9&1)&+!*6!;0!<%=!+&.&'+9%!7+*e&9).>!

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)%&! 6-+.)!.)&7!9*1.-.).! -1!)%&!removal of the 5’ leader sequence of precursors tRNAs!50!

OP'.&!<>!$%-.!&..&1)-'/!'9)-3-)0!-.!9*1.&+3&4!-1!'//!4*;'-1.!*6!/-6&!I?*77&+!&)!'/>8!"_U_M>!

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&1@0;&!IOP<M!'14!'!7+*)&-1J*1/0!&1@0;&!I'M>!$%&!.)+(9)(+&!'14!;*4&!*6!'9)-*1!*6!5*)%!

)07&.! *6! &1@0;&.! %'3&! 5&&1! .)(4-&4! -1! 4&)'-/.! I5M! .%*,-1:! '1! -1)&+&.)-1:! 9'.&! *6!

9*13&+:&1)!&3*/()-*1!5&),&&1!OP<!'14!7+*)&-1J*1/0!OP'.&!<>!A-1'//08! )%&!4-3&+.-)0!*6!

.(5.)+')&.! '14! *6! 6(19)-*1.! %&/4! 50! )%&! 4-66&+&1)! 2-14.! *6! OP'.&! <! &1@0;&.! -1! 5*)%!

Z(2'+0*)&.!'14!7+*2'+0*)&.!,-//!5&!4-.9(..&4!I9M>!

!

#? @;#).*2*()*%3.*"$(7.4)#:*.$LD&.*%3#%*<#%#:D).)*removal of the 5’ leader sequence of precursorN%@;8),*G3()*.$LD&.*.O()%)*#)*0/%3*#*@(0/$"<:./-4/%.($*#$9*#*-4/%.($N/$:D*B/4&*

!

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activity that by its cleavage “removes all extra nucleotides present at the 5’ terminus of

the precursor (..) exposing the 5’ end of the mature tRNA” (Robertson et al.8!Ubg"M>! L1!

Ubf#8!OP'.&!<!,'.!-4&1)-6-&4!'.!'!+-5*@0;&!I,%-9%!.)'14!6*+!02/%1(9/&-9!'9-4!&1cD+(M>!

L14&&48! S-41&0! Q/);'1! '14! 9*//&':(&.! .%*,&4! )%')! -1! E,* </:(! '14!P,* )"0%(:(), “the RNA

;*-&)-&.! I>>M! 9'1! 9/&'3&! )OPQ!7+&9(+.*+!;*/&9(/&.! I>>M>!$%&!OPQ!'9).!'.! '! )+(&! 9')'/0.)!

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catalytic activities” (GuerrierJ$'2'4'! &)! '/>8! Ubf#M>! $%-.! 6*+;! *6! OP'.&! <! ,'.! ,-4&/0!

9*1.-4&+&4!'.! )%&! .*/&! 6*+;!*6!OP'.&!<!(1)-/! '!4&9'4&!':*! IQ/);'11!"__gM>! L14&&48! -1!

Q+9%'&'8! C'9)&+-'! '14! Z(2'+0*)&.8! '//! OP'.&! <! 9%'+'9)&+-@&4! ,&+&8! (1)-/! "__f8!

+-5*1(9/&*7+*)&-1!IOP<M!7'+)-9/&.!9*1)'-1-1:!'!+-5*@0;&>!$%-.!6-14-1:!*6!'!9*1.&+3&4!OPQ!

%*/4-1:!)%&!9')'/0)-9!'9)-3-)0!*6!)%&!OP<!7'+)-9/&.!-1!)%&!)%+&&!4*;'-1.!*6!/-6&!/&4!)*!)%&!

! Tb!

%07*)%&.-.!)%')!OP'.&!<!,*(/4!5&!*1&!*6!)%&!.*/&!3&.)-:&!*6!)%&!7+*7*.&4!7+&J5-*)-9!“RNA

world” (a term coined by Gilbert in 1986) and more controversially, to the assumption

)%')!OP'.&!<!,*(/4!.*/&/0!'14!(1-3&+.'//0!*99(+!'.!'1!OP<!6*+;>!!

$%&!*99(++&19&!*6!'!1*3&/!6*+;!*6!7+*)&-1J*1/0!OP'.&!<!,'.!6-+.)!6*+;'//0!4&;*1.)+')&4!

-1!"__f!50!?*/@;'11!&)!'/>!I"__fM!)%')!9%'+'9)&+-@&4!%(;'1!;-)*9%*14+-'/!OP'.&!<>!$%-.!

&1@0;&!-.!9*;7*.&4!*6!#!7+*)&-1.!IGO<<U8!"!'14!#M8!,-)%!GO<<#!I/')&+!9'//&4!<OYO<!6*+!

“<O*)&-1JY1/0!OP'.&!<”)!%*/4-1:!)%&!9')'/0)-9!'9)-3-)0>!L1!6'9)8!-)!,'.!'/+&'40!.(.7&9)&4!

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9*;7'+);&1).! *1! '! 9*1.&+3&4! 9')'/0)-9! OP'.&! <! OPQ! I.&&! ?'+);'11! &)! '/>8! "__bM>! L1!

7'+)-9(/'+8! ),*!&'+/0!'+)-9/&.!4')-1:! 6+*;!Ubb^!'14!"___!50!$%*;'.!&)!'/8!%'4!'/+&'40!

.(::&.)&4!)%')!-1!S7-1'9%!I'-($#<(#*/:.4#<.#M!9%/*+*7/'.)8!OP'.&!<!'9)-3-)0!,'.!7()')-3&/0!

%&/4!50!'!.-1:/&!7+*)&-1!I$%*;'.!&)!'/>8!Ubb^M!'nd that furthermore, this enzyme did “not

()-/-@&!)%&!+-5*@0;&J)07&!7+&JtRNA cleavage mechanism” (Thomas et al., 2000). ?*,&3&+8!

)%&.&!+&.(/).!,&+&!+&9&-3&4!,-)%!.9&7)-9-.;!'.!)%&!1')(+&!*6!)%&!7+*)&-1!'14!-).!9*4-1:!

:&1&!9*(/4!1*)!5&! -4&1)-6-&4>!P*1&)%&/&..8!,-)%! )%&!'43&1)!*6! )%&! 9'7'9-)0! )*! .&R(&19&!

;'..-3&/0! =PQ! '14! )%&! -;7*+)'1)! &66*+).! )*! .&R(&19&! 9*;7/&)&! :&1*;&.! 6+*;! '! 3'.)!

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6*+;!*6!)%-.!&1@0;&!5&9';&!%-:%/0!7/'(.-5/&!I<-12&+!&)!'/>8!"_U#M>!

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%*.)!/'5*+')*+0!I\*5&+)!&)!'/>!"_U_j!\();'11!&)!'/>!"_U"M>!L1!5+-&68!Q+'5-4*7.-.!&19*4&.!

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9%/*+*7/'.).!,%-/&!<OYO<"! '14!#!7&+6*+;!1(9/&'+!OP'.&! <! '9)-3-)0>!Q99*+4-1:/08!OP<!

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OP'.&!<!,&+&!)%&1!6(+)%&+!9%'+'9)&+-.&4!-1!'!/'+:&+!3'+-&)0!*6!*+:'1-.;.8!-1!G4D-#$/)/&#*

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IC*11'+4!&)!'/>8!"_U[M>!L1!"_U^8!'!7%0/*:&1&)-9!'1'/0.-.!ID&9%1&+!&)!'/>8!"_U^M!.%*,&4!)%')!

<OYO<!*99(+.!-1!T!*()!*6!)%&!^!;'-1!&(2'+0*)-9!7%0/'!'14!)%')!<OYO<!'14!OP<!OP'.&!<!

! ^_!

'+&!;()('//0!&B9/(.-3&8!-1!&1)-+&!*+:'1-.;.!*+!-1!9*;7'+);&1).!d!*+:'1&//&.!IA-:(+&!U[M>!

L1! /'14!7/'1).! )%')!9*1.)-)()&!*(+!;'-1!;*4&/.8!*1/0!)%&!7+*)&-1J*1/0! 6*+;!*6!OP'.&!<!

*99(+.!'14!OP<!OP'.&!<!,'.!&1)-+&/0!/*.)>!!

K&+0!+&9&1)/08!5&0*14!)%&!*99(++&19&!*6!<OYO<!-1!Z(2'+0*)&.8!)%&!4-3&+.-)0!*6!OP'.&!<!-1!

)%&! 4-66&+&1)! 4*;'-1.! *6! /-6&! ,'.! 9%'//&1:&4! ':'-1>! $%&! :+*(7! *6! O*/'14! ?'+);'11!

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proteins “?QO<”! I?*;*/*:.! *6! QR(-6&B! OP'.&! <M>! ?QO<! ,'.! 9%'+'9)&+-.&4! -1! '!

%07&+)%&+;*7%-/-9! 5'9)&+-(;! 8Q"(B.O* #./:(<")>! L)! -.! )%&! .;'//&.)! 7+*)&-1J*1/0! 6*+;! *6!

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4*;'-1!*1/0!I,%-/&!<OYO<!'+&!(.('//0!'+*(14![_2='M>!L)!%'.!)%&1!5&&1!.%*,1!)%')!)%-.!

6';-/0! *6! 7+*)&-1.! -.! 4-.)+-5()&4! -1! 5*)%! C'9)&+-'! '14! Q+9%'&'>! ?*,&3&+8! )%-.! 6*+;! *6!

7+*)&-1!*1/0!OP'.&! <! *6)&1! 9*J&B-.).!,-)%! )%&!OP<! 6*+;!*6!OP'.&! <>! Q.! .(9%8! -)! -.! 0&)!

(19/&'+!,%')! -.! )%&!&B'9)! 6(19)-*1!*6!?QO<!'14!,%&)%&+!OP<!OP'.&!<!'14!?QO<!%'3&!

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:3,(023'.!!!!!

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! [[!

>BBBO =3",(023'.-,032$-.&

#? E)<3.4(<3(#*</:(W*!E,*</:(!-.!'!\+';J1&:')-3&!C'9)&+-'!9*;;*1/0!6*(14!-1!)%&!;-9+*5-*)'!*6!;';;'/.>!L)!%'.!5&&1!(.&4!.-19&!4&9'4&.!-1!;-9+*5-*/*:0!'14!;*/&9(/'+!5-*/*:0>!`&0!6-14-1:.!-1!5-*/*:0!%'3&!5&&1!;'4&!(.-1:!)%-.!;*4&/!*+:'1-.;!/-2&!)%&!4-.9*3&+0!*6!*7&+*1.!Im'9*5!&)!'/>8!Ub[_!'14!Ub[UM8!)%&!'9%-&3&;&1)!*6!:&1&)-9'//0!;*4-6-&4!*+:'1-.;.!IH*%&1!&)!'/8!Ubg#M8!%*,!=PQ!+&7/-9')&.!ID&%;'1!&)!'/>8!Ub^fM!'14!%*,!)%&!:&1&)-9!9*4&!6(19)-*1.!IH+-92!'14!'/>8!Ub[UM>!E,*</:(!.(5.)+'-1.!(.&4!-1!5-*/*:0!/'5*+')*+-&.!'//!*+-:-1')&!6+*;!),*!.)+'-1.!9'//&4!`JU"!and B. For example, the two strains DH5α and DH10B (that was branded under the name $Y<U_! 50! $%&+;*A-.9%&+dL13-)+*:&1M! 5*)%! 4&+-3&! 6+*;! )%&! `JU"! .)+'-1>! $%&! CD"U!.(5.)+'-1!4&+-3&.!6+*;!)%&!C!.)+'-1>!!!S)+'-1.!(.&4!6*+!7/'.;-4!';7/-6-9')-*1N!!

· DH5α strain:!

$%&.&!E,*</:(!9&//.!,&+&!&1:-1&&+&4!)*!-19+&'.&!)+'1.6*+;')-*1!&66-9-&190>!$%&0!'+&!`Y!6*+!&14*1(9/&'.&!L! I.$98IM!'14!4&6-9-&1)! 6*+!%*;*/*:*(.!+&9*;5-1')-*1!I4.<8IM>!$%&0!'+&!9*1.-4&+&4!'.!(.&6(/!)*!9/*1&!/'+:&!7/'.;-4.>!\&1*)07&N!=–*.$98I*A:$MXX*%3(N1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15

Δ(lacZYAN#4A=?YISUC*3)9@IZV4[–mK+), λ–!!

· $Y<U_!.)+'-1>!

$Y<U_! 9&//.! '+&! 5+'14&4! 50! $%&+;*A-.9%&+>! $%&0! '+&! 7+*5'5/0! -4&1)-9'/! )*! =?U_C!IL13-)+*:&1M!'14!PZC!U_J5&)'>!$%&0!'+&!'/.*!+&9*;5-1')-*1!'14!&14*1(9/&'.&!L!4&6-9-&1)!I+&9QU! d! &14QUM>! H%&;-9'//0! 9*;7&)&1)! $Y<UY! 9&//.!,&+&! +*()-1&/0! (.&4! 6*+! 7/'.;-4!';7/-6-9')-*1>!\&1*)07&N! )%4,* [NIT* =–* Δ(araNleu)7697[Δ(rapA'Ncra' )] Δ(lac)X74[Δ('yahHN&3-E?\*9"-:(<#%(/$V]IX^XINSTZS_I?`$&-1NA:%6\* A#:[IS* A#:EI]* .IX–V(<9aG* &<48?*φ80dlacZΔM15 recA1 relA1 endA1 Tn10.10 nupG rpsL150(StrR) rph+ spoT1 Δ(mrrN3)9@H'NmcrBC) λ–*H()).$).V9$#8*A:&'*A:D!*:-O[*&4.1*&"48?*;/$).$).V<3(8*A#%b*B3"8c*D(A8*DA<d?*=4#&.)3(B%VB:31*&A:8*B4"P?!!!!S)+'-1.!(.&4!6*+!7+*)&-1!7+*4(9)-*1N!!

· CD"UI=Z#M!.)+'-1N!

$%&.&!9%&;-9'//0!9*;7&)&1)!9&//.!9*1)'-1!)%&!$g!OPQ!7*/0;&+'.&!:&1&!-1!6(.-*1!,-)%!)%&!L<$\J-14(9-5/&!7+*;*)&+>!$%&0!'+&!(.&4!)*!&B7+&..!7/'.;-4.!,-)%!)%&!$g!7+*;*)&+>!\&1*)07&N!E,*</:(*)%4,*P*=–*/&-G*A#:*9<&*:/$*3)9'PV4P–&P–) λ(DE3 [lacI lacUV5NGZ-_Z*($9I*)#&Z*$($]\?*`&#:Pe\[N12(λS)*!

! [g!

· O*.&))'"I=Z#M!7D0.S!.)+'-1N!!

$%&.&! .)+'-1.! 4&+-3&! 6+*;! CD"U>! $%&0!,&+&! 4&.-:1&4! )*! &1%'19&! &(2'+0*)-9! 7+*)&-1.!&B7+&..-*1>!A*+!)%-.!7(+7*.&8!)%&0!,&+&! .(77/&;&1)&4!,-)%!)%&!+'+&!9*4*1.!Q\\8!Q\Q8!QEQ8!HEQ8!HHH!'14!\\Q>!$%&.&!)OPQ!:&1&.!'+&!9'++-&4!50!'!9%/*+';7%&1-9*/!+&.-.)'1)!7/'.;-4>!\&1*)07&N!E,*</:(*)%4,*P*=–*/&-G*A#:*9<&*:/$c*3)9'PV4P–&P–) λ(DE3 [lacI lacUV5NGZ-_Z*($9I*)#&Z*$($]\?*`&#:Pe\[N12(λS) pLD)'@8@E`GZ-T_*(:.J*#4AY*%34Y*%D4Y*A:DG*%34G*#4Aa*&.%G*:."a*-4/R*/4(-I]8\V1&@?*!!

0? 8A4/0#<%.4("&*%"&.B#<(.$)W*!

· \K#U_U!

$%-.! .)+'-1! %'.! '! H^f! 9%+*;*.*;'/! 5'92:+*(14! I,-)%! O-6';7-9-1! +&.-.)'19&M>! $%&! $-!7/'.;-4!7G<b_!%'.!:&1)';09-1&!+&.-.)'19&>!$J=PQ!.&R(&19&!,'.!+&;*3&4!6+*;!)%&!$-!7/'.;-4>!Q.!.(9%8!*1/0!)+'1.6*+;')-*1!,-)%!'!5-1'+0!3&9)*+!9*1)'-1-1:!)%&!$-J+&:-*1!,-//!:-3&!'!$J=PQ!5-1'+0!.0.)&;!'//*,-1:!)+'1.6&+!*6!=PQ!-1)*!)%&!:&1*;&!*6!)%&!)+'1.6*+;&4!7/'1)>!$%&!\K#U_U!.)+'-1!%'.!\&1)';09-1!'14!O-6';7-9-1!+&.-.)'19&>!Q!)%-+4!'1)-5-*)-9!-.!(.&4!6*+!)%&!5-1'+0!3&9)*+>!!!

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#? =/4*21@*<:/$($AW*!

· 7\ZGiJ$!&'.0!I<+*;&:'MN!!

$%-.! 7+&Jlinearized vector contains 3’J! $! *3&+%'1:! ')! )%&! -1.&+)-*1! .-)&>! $g! 7+*;*)&+!+&:-*1.!6/'12!)%&!;(/)-7/&!9/*1-1:!.-)&!IGHSM>!$%&!GHS!+&:-*1!-.!-1.&+)&4!-1!)%&!7&7)-4&!coding region for βJ:'/'9)*.-4'.&>!Q//*,-1:!5/(&J,%-)&!.9+&&1!.&/&9)-*1!*6! )+'1.6*+;&4!9/*1&.>!L1.&+)&4!=PQ!9'1!5&!+&;*3&4!50!'!.-1:/&!+&.)+-9)-*1!&1@0;&!4-:&.)-*1!,-)%!C.)WL8!Z9*OL!*+!P*)L>!!!!!

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· 7=YPYO!"_g!

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· 7Z'+/&0\')&!

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· 7Z$"fC!6*+!Q)<OYO<"!'14!Q)$OW#!&B7+&..-*1!

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! .

! [b!

;02+(0.B5. G($(.B5. ;02+(0.-(NL($"(.

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)%&!.';7/&!,'.!3*+)&B&4>!$(5&.!,&+&!9&1)+-6(:&4!^!;-1()&.!')!U[!___!:!ITkHM>!$%&!(77&+!'R(&*(.! 7%'.&! 9*1)'-1-1:! )%&! OPQ.! ,'.! )+'1.6&++&4! )*! '! 1&,! )(5&! '14! "^_! v/! *6!-.*7+*7'1*/!I_8^!;/dU;D!)+-@*/M!,'.!'44&4>!Q6)&+!3*+)&B-1:!'14!5’ -19(5')-*1!')!O$8!)(5&.!,&+&! 9&1)+-6(:&4! f! ;-1()&.! ')! U[!___! :! ITkHM>! S(7&+1')'1)! ,'.! +&;*3&4! '14! 7&//&)!,'.%&4!,-)%!^__!v/!*6!&)%'1*/!g_n>!Q6)&+!9&1)+-6(:')-*1!')!U[!___!:!ITkHM!6*+!^!;-18!)%&!.(7&+1')'1)!,'.!+&;*3&4>!$*!&1.(+&!7+*7&+!+&;*3'/!*6!+&.-4('/!&)%'1*/8!'!.%*+)!.7-1!*6!^!.&9*14.!,'.!7&+6*+;&4!'14!)%&!+&;'-1-1:!/-R(-4!,'.!+&;*3&4>!$%&!7&//&)!,'.!'-+J4+-&4!5&6*+&!5&-1:!+&.(.7&14&4!-1!"_!v/!*6!.)&+-/&!,')&+>!!

0? F;#).*%4.#%&.$%W*!=P'.&!)+&');&1)!,'.!7&+6*+;&4!(.-1:!)%&!=P'.&!L!&1@0;&>!O&'9)-*1!,'.!7&+6*+;&4!-1!'!)*)'/!3*/(;&!*6!U__!v/>!U_!v/!*6!=P'.&!L!c!G:H/"!IU_X!5(66&+M!,'.!'44&4!)*!U!)*!U_!v:!*6!OPQ>!=P'.&!L!I$%&+;*A-.%&+M!,'.!'44&4! -1!'! +')-*!*6!UE!7&+!U!v:!*6!OPQ!,-)%!.)&+-/&!,')&+! )*! +&'9%!U__!v/>!O&'9)-*1!,'.! -19(5')&4!#_!;-1()&.!')!#gkH!'14!.)*77&4!(.-1:!7%&1*/d9%/*+*6*+;!&B)+'9)-*1>!!

<? 8A#4/).*A.:*%/*.$)"4.*@;8*($%.A4(%DW*!$*!9%&92!6*+!'10!OPQ!4&:+'4')-*18!)%&!(.('/!;&)%*4!-.!)*!+(1!U__!1:!*6!7(+-6-&4!OPQ!*1!'!6*+;'/4&%04&!4&1')(+-1:!:&/>!?*,&3&+!)%-.!7+*9&..!-.!)-;&!9*1.(;-1:!'14!&13-+*1;&1)!1*1J6+-&14/0>!A*+!+*()-1&!9%&92!*6!OPQ!-1)&:+-)08!'!.-;7/&!1*1!4&1')(+-1:!':'+*.&!:&/!9'1!5&! used. 100 ng of RNA is simply added to 1 µl of “OPQ! .)*7” solution (40%(v/v

6*+;';-4&8!U_!;G!Z=$Q8!_>__"^n!5+*;*7%&1*/!5/(&8!_>__"^n!B0/&1&!90'1*/M!/*'4-1:!5(66&+!'14!&/&9)+*7%*+&.-.!-.!9'++-&4!*()!')!U__!K!6*+!U^!;-1>!Q6)&+!3-.('/-@')-*1!(14&+!EK!/-:%)8!OPQ.!'+&!9*1.-4&+&4!'.!1*1J4&:+'4&4!-6!),*!4-.9+&)&!5'14.!9'1!5&!.&&1!6*+!)%&!UfS!'14!"fS!+-5*.*;'/!OPQ.8!,-)%!1*!.;&'+>!

.

9? d.:*-"4(B(<#%(/$*/B*@;8W*!$*!&B)+'9)!OPQ!6+*;!'!4&1')(+-1:!:&/8!,&!(.&4!)%&!7+*)*9*/!*6!$>]>P-/.&18!"_U#>!C+-&6/08!)%&! 5'14! *6! -1)&+&.)! -.! +&;*3&4! 6+*;! )%&! :&/! (.-1:! '! +'@*+! 5/'4&! '14! 7/'9&4! -1! '!;-9+*9&1)+-6(:&!)(5&!,-)%!T__!v/!*6!:&/!&/()-*1!5(66&+!I"_!;G!$+-.J?H/!7?!g>^8!_8"^!G!S*4-(;!'9&)')&8!U!;G!Z=$Q8!_8"^n!S=SM>!Q6)&+!6+&&@-1:!6*+!U^!;-1!')!Jf_kH8!)%&!OPQ!-.!/&6)!)*!4-66(.&!*3&+1-:%)!50!/&'3-1:!)%&!)(5&!')!+**;!)&;7&+')(+&>!Q6)&+!9&1)+-6(:')-*1!')!+**;!)&;7&+')(+&!6*+!U_!;-1!')!;'B-;(;!.7&&48!)%&!.(7&+1')'1)!-.!)+'1.6&++&4!)*!'!1&,!)(5&!'14!OPQ!-.!+&)+-&3&4!(.-1:!7%&1*/J9%/*+*6*+;!6*//*,&4!50!&)%'1*/!7+&9-7-)')-*1>!!!

! f_!

.? @.7.4).*G4#$)<4(-%(/$W*!9=PQ.!.01)%&.-.!,'.!7&+6*+;&4!(.-1:!7(+-6-&4!OPQ.8!'6)&+!=PQ!+&;*3'/!(.-1:!=P'.&!L!I$%&+;*A-.%&+M>!A*+!'//!7(+7*.&.!IO$JqPCR, 3’ or 5’ RACE), an equal quantity of RNA was

(.&4!6*+!'//!.';7/&.>!O&3&+.&!$+'1.9+-7)-*1!,'.!7&+6*+;&4!(.-1:!&-)%&+!S(7&+.9+-7)!LK!O$!I$%&+;*A-.%&+M! *+! GoScript™ Reverse Transcriptase! I<+*;&:'M>! L1! &'9%! 9'.&! )%&!supplier’s instructions were followed. To achieve global reverse transcription of RNAs a

;-B!*6!*/-:*!4$!7+-;&+.!'14!+'14*;!%&B';&+!7+-;&+.!,'.!(.&4>!A*+!+&3&+.&!)+'1.9+-7)-*1!of specific RNAs (for example in 3’ and 5’ race) a specific primer was used.!!

B? @GNQ21@W*!O$JR<HO!-.!'!;&)%*4!(.&4!)*!4&)&+;-1&!)%&!+&/')-3&!/&3&/!*6!&B7+&..-*1!*6!:-3&1!:&1&.>!$%&!';*(1)!*6!9=PQ.!-.!;&'.(+&4!50!)%&!1(;5&+!*6!909/&!1&9&..'+0!)*!+&'9%!'!9+*..-1:!)%+&.%*/4!IH)M8!4&6-1&4!'.!'!:-3&1!';*(1)!*6!6/(*+&.9&19&!;&'.(+&4>!C0!9*;7'+-1:!)*!)%&!relative level of “housekeeping” genes (various endogenous controls were used, see

7+-;&+.!/-.)!6*+!4&)'-/.M8!)%&!+&/')-3&!&B7+&..-*1!/&3&/!*6!4-66&+&1)!:&1&.!9'1!5&!9*;7'+&4!5&),&&1!.';7/&.>!C+-&6/0!9=PQ.!,&+&!4-/()&4!U_!6*/4!'14!;-B&4!,-)%!"^_!;G!*6!.&1.&!'14!'1)-.&1.&!7+-;&+.!'14!^!v/!*6!Eurogentec Takyon™ SYBR® 2X qPCR Mastermix Blue.

O&'9)-*1.! ,&+&! 7&+6*+;&4! ,-)%! )%+&&! )&9%1-9'/! +&7/-9')&.! -1! #fTJ,&//! 7/')&.! ,-)%! '!D-:%)H09/&+! Tf_! O&'/J$-;&! <HO! S0.)&;! IO*9%&M>! <HO! 7+*:+';! ,'.! '.! 6*//*,! I-1-)-'/!4&1')(+')-*1!.)&7!*6!^!;-1()&.!6*//*,&4!50!T_!909/&.!*6!U_!.&9*14.!')!b^kH8!U^!.&9*14.!')![_kH!'14!U^!.&9*14.!')!g"kHM>!<HO!&66-9-&19-&.!,&+&!9'/9(/')&4!6*+!'//!+&'9)-*1.>!Y1/0!<HO!&66-9-&19-&.!.(7&+-*+!)*!_8b!,&+&!+&)'-1&4>!Q6)&+!&1.(+-1:!)%')!'//!)&9%1-9'/!+&7/-9')&.!%'3&!/&..! )%'1! _8^! H)! *6! 4-66&+&19&8! )%&! +&/')-3&! &B7+&..-*1! -.! 9'/9(/')&4! (.-1:! )%&! 4&/)'! H)!;&)%*4>!!

A? <@GN21@*!H/&'3&4!OPQ!,'.!&B)+'9)&4!6+*;!:&/!(.-1:!)%&!;&)%*4!4&.9+-5&4!50!$>]>P-/.&18!"_U#>!$%&1!U!v:!*6!RNA was ligated using T4 RNA ligase (NEB) according to manufacturers’

-1.)+(9)-*1.>!O&'9)-*1!,'.!9'++-&4!')!#gkH!6*+!U!%*(+!'14!&1@0;&!%&')!-1'9)-3')&4!')![^kH!6*+!U^!;-1>!O&3&+.&!)+'1.9+-7)-*18!(.-1:!9-+9(/'+-.&4!OPQ!'.!'!;')+-B!,'.!7&+6*+;&4!(.-1:!Superscript IV (Thermo fisher) following manufacturers’ instructions.!Q!.7&9-6-9!7+-;&+!,'.!4&.-:1&4!)*!;')9%!)%&!&B7&9)&4!9-+9(/'+-.&4!OPQ!.&R(&19&>!$%&1!1&.)&4!<HO!,'.!7&+6*+;&4!(.-1:!Uv/!*6!9=PQ!'.!'!)&;7/')&>!$%&!6-1'/!<HO!+&'9)-*1!,'.!.(5;-))&4!)*!<HO!9/&'1J(7! (.-1:! )%&!G'9%&+&0!P':&/! <HO! 9/&'1J(7! 2-)>! QJ)'-//-1:!,'.! 7&+6*+;&4! *1! )%&!&/()&4!=PQ!(.-1:!\*!$'R!<*/0;&+'.&! I<+*;&:'M!'14!=PQ!,'.! )%&1! -1.&+)&4! -1!7\ZG!I<+*;&:'M! '99*+4-1:! )*!;'1(6'9)(+&+.’ instructions. Insert was sequenced using M13

7+-;&+.!50!S'1:&+!.&R(&19-1:>!!!

! fU!

3? 3’ and 5’ 4#-(9*#&-:(B(<#%(/$*/B*<F;8*.$9)*V@81E?W*!3’RACEN!!3’ RACE was performed by adapting the protocol of Scheer et al., 2020. Total RNA

&B)+'9)-*1!'14!=P'.&!)+&');&1)!,'.!7&+6*+;&4!'.!-14-9')&4!'5*3&>!Q6)&+!OPQ!-1)&:+-)0!,'.!9%&92&4!*1!'1!':'+*.&!:&/8!^!v:!*6!7(+-6-&4!OPQ!,'.! /-:')&4!)*!U!v/!*6!U_!vG!O"U!'47')*+>!$%-.!7+-;&+!<+-;&+!-.!5’adenylated (5’rAppM!'14!'.!.(9%!9'1!'9)!'.!.(5.)+')&.!6*+!$T! /-:'.&.! -1! )%&! '5.&19&! *6! Q$<8! 7+&3&1)-1:! 6+*;! '4&10/')-*1! *6! (1,'1)&4! 7+*4(9)!I+OPQ8!)OPQ!'14!;-OPQM>!Q44-)-*1'//0!)%-.!primer is blocked in 3’ by a dideoxycytosine,

7+&3&1)-1:! )%&! 6*+;')-*1! *6! 7+-;&+! 9*19')&;&+>! <+-;&+! .&R(&19&! -.!d^+Q77dH$\QHPPPPPPPPPPPPPPP$\\QQ$$H$H\\\$\HHQQ\\Hd#44Hd!]')&+!,'.! '44&4! )*! '! 3*/(;&! *6! TT! v/! '14! )%&! +&'9)-*1!,'.! -19(5')&4! ')! [^kH! 6*+! #!;-1()&.!'14!)%&1!7()!*1!-9&!6*+!"!;-1()&.>!^v/!*6!$T!OPQ!/-:'.&!5(66&+!IPZCM!,'.!'44&4!'.!,&//!'.!U!v/!*6!$T!OPQ!/-:'.&!U!I..OPQ8!PZCM>!Q6)&+!-19(5')-*1!6*+!U!%*(+!')!#gkH8!)%&!/-:')&4!OPQ!7+*4(9).!,&+&!7(+-6-&4!(.-1:!)%&!G'9%&/&0!P':&/!P(9/&*.7-1!OPQ!7/'1)!2-)!"8^!v:!*6!/-:')&4!OPQ!,'.!(.&4!6*+!+&3&+.&!)+'1.9+-7)-*1!IO$M>!O$!,'.!7&+6*+;&4!(.-1:!SSIV (Thermo) according to the manufacturer’s instruction. For this reaction, a RT short

7+-;&+! IQUT[gM! ,'.! (.&48! '//*,-1:! *1/0! ';7/-6-9')-*1! *6! '4'7)&+J/-:')&4! OPQ.>!Q;7/-6-9')-*1!,'.!7&+6*+;&4!')!^[kH8!,%-9%! -.! )%&!;'B-;'/! )&;7&+')(+&!')!,%-9%! )%&!&1@0;&!%'.!U__n!&66-9-&1908!'99*+4-1:!)*!)%&!;'1(6'9)(+&+>!Q6)&+!O$8!OP'.&!?!,'.!'44&4!IUv/!d!+&'9)-*1M>!$%&!+&.(/)-1:!9=PQ.!*6!),*!O$!+&'9)-*1!d!.';7/&!,&+&!7**/&48!.(5e&9)&4!)*!7%&1*/J9%/*+*6*+;!'14!U!v/!,'.!(.&4!'.!)&;7/')&!6*+!1&.)&4!<HO>! A-1'/! <HO!7+*4(9).!,&+&! 7(+-6-&4! (.-1:! QG<(+&! 5&'4.! IQ:&19*(+)M>! =PQ!R('1)-)0!,'.!4&)&+;-1&4!(.-1:!l(5-)i!'14!7+*6-/&!,'.!9%&92&+!(.-1:!'!C-*'1'/0&+i!9%-7>!Q6)&+!/-5+'-+0!7+&7'+')-*18!7(+-6-&4!<HO!7+*4(9).!,&+&!.&R(&19&4!,-)%!)%&!L//(;-1'!G-S&R!.0.)&;>!!5’ RACEN!!$*)'/!OPQ!&B)+'9)-*18!=P'.&!)+&');&1)!'14!'4'7)*+!/-:')-*1!,'.!7&+6*+;&4!'.!-14-9')&4!for 3’ RACE. As we were investigating the termini of a cleaved RNA, no dephosphorylation

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&.)'5/-.%&4>!G0!)%&.-.!,*+2!'-;&4!')!9*1)+-5()-1:!)*!'1.,&+!)%&.&!R(&.)-*1.>!

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1(9/&'+! <OYO<"! OPQ! .(5.)+')&.! -.! '44+&..&4>! L1-)-'//08! ,&! %'4! &13-.':&4! )*! /**2! 6*+!

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7+&.&1)! -1! '1! -1)+*1! *6! )%&! 7+&J;OPQ! &19*4-1:! GQAU8! )%&! ;'-1! +&7+&..*+! *6! OPQ!

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MAF1 gene. They hypothesized that this conserved intronic region was “unlikely an

alternative splicing exon” as it could not be found in MAF1 mature mRNA in all plant

)+'1.9+-7).!.)(4-&48!5()!'!1&,!19OPQ>!?*,&3&+8!)%&0!9*(/4!1*)!4&)&9)!-)!50!'!1*+)%&+1!5/*)!

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9*1.&+3&4!)OPQJ/-2&!.)+(9)(+&!I$DSM>!$%&!$DS!7+&.&1)!-1!84#0(9/-)()*%3#:(#$#!GQAU!:&1&!

IA-:(+&!"^Q8!CM!9'1!5&!;*4&/&4!-1!"=!'14!#=!(.-1:!OPQ6*/4!.*6),'+&!+&3&'/-1:!'!.)+(9)(+&!

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(At5g1320) gene is depicted with its 5’!and 3’ UTR region as grey boxes, exons as black boxes and

-1)+*1.! '+&! +&7+&.&1)&4! 50! /-1&.! 50! /-1&.>! S9'/&! 5'+! +&7+&.&1).! U__! 1(9/&*)-4&.! IQM>! $%&! gT!

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"=! .)+(9)(+&! *6! <%&10/'/'1-1&! )OPQ! 6+*;! V&'.)! IS,* <.4.7()(#.! 6+*;! /'5*+')*+0! .)+'-1! S"ffHM! -.!

.%*,1! )*! %-:%/-:%)! )%&! +&.&;5/'19&! 5&),&&1! )%&! 7+&4-9)&4!"=! .)+(9)(+&! *6! Q)GQAU!$DS!'14! '!

“canonical” tRNA (C). #=!.)+(9)(+&.!*6!)%&!9*1.&+3&4!-1)+*1-9!)OPQ!/-2&!S)+(9)(+&!*6!Q+'5-4*7.-.!

GQAU!I/&6)M!'14!*6!<%&10/'/'1-1&! )OPQ! 6+*;!V&'.)! IS,* <.4.7()(#.! 6+*;! /'5*+')*+0!.)+'-1!S"ffHM!

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U#!'14!"_!5'.&.!(7.)+&';!*6!)%&!$DS!.)'+)!.-)&>!E.-1:!OPQ6*/48!,&!6*(14!)%')!)%-.!OPQ!

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)+'1.9+-5&.!)OPQ!'14!*)%&+!19OPQ!I^S!+-5*.*;'/!OPQ8!E[!.7/-9&*.*;'/!OPQ!'14!gSD!6*+!

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cleave 5’ leader sequences of precursors tRNA and of a tRNAJ.1*OPQ!7+&9(+.*+!($*7(7/!

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9*(/4!9/&'3&!Q)GQAU!$DS!($*7(%4/*'14!($*7(7/>!Q/.*!,&!,'1)&4!)*!4&)&+;-1&!-6!)%-.!'9)-3-)0!

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-1!]$!IH*/JYM!'14!-4/4-^! 21*92J*()! I`YM!5'92:+*(14>!L;':&.!,&+&! )'2&1!Uf!4'0.!'6)&+!KL\S! -16&9)-*1>!

</'1).!,&+&!9*1.&R(&1)/0!"f!4'0.!'6)&+!:&+;-1')-*1!IS)':&!^!6+*;!C*0/&.!'14!'/>M>!Q//!7/'1).!,&+&!.*,1!')!

)%&!.';&!)-;&!'14!:+*,1!-1!)%&!.';&!9*14-)-*1.>!]$!a!,-/4!)07&8!7"!a!<OYO<"8!7#!a!7+*+7#8!ZK!a!&;7)0!

3&9)*+8!74.!a!7%0)*&1&!4&.')(+'.&>!

!!

! UU_!

!

F2*L0(.6TN!8(P0(-($,3,21(.2+3*(-.%W.P'3$,-.6.&((#-.3W,(0.>BG9.2$W(",2%$.3$).2,-.(WW(",.3,.,R(."(''L'30.

'(1('>!O&7+&.&1)')-3&!7%*)*:+'7%.!*6!7/'1).!#!,&&2.!'6)&+!KL\S!-16&9)-*1!':'-1.)!<OYO<"!I'5*3&M!*+!,-)%!

'1! &;7)0! 3&9)*+! I5&/*,M! '+&! .%*,1! I/&6)M>! C/'92! 5*B&.! *1! /&'3&.! -14-9')&.!,%&+&! '! _>^!;;! 5'14!,'.!

4-..&9)&4!-1!)%&!/&'6!;-4+-5!'14!6(+)%&+!7+*9&..&4!)*!5&!*5.&+3&4!50!)+'1.;-..-*1!&/&9)+*1-9!;-9+*.9*7&!

I$ZGM>!O&7+&.&1)')-3&!-;':&.!'6)&+!3-.('/-.')-*1!*1!'!?-)'9%-!?!g^__!)+'1.;-..-*1!&/&9)+*1!;-9+*.9*7&!'+&!

.%*,1!6*+!KL\S!':'-1.)!<OYO<"!I(77&+!'14!;-44/&M!*+!6*+!)%&!&;7)0!3&9)*+!I/*,&+M!*1!)%&!+-:%)>!S9'/&!5'+!

-.!-4&1)-9'/!6*+!)%&!)%+&&!$ZG!-;':&.>!H&//(/'+!9*;7'+);&1).!'+&!-14-9')&4!(.-1:!5/'92!'++*,.>!

!

! !

! UUU!

$%-.!+&.(/)!1-9&/0!9*;7/&;&1).!)%&!*1&!7+&3-*(./0!*5)'-1&4!-1!)%&!+&.&'+9%!:+*(7>!L14&&48!

\();'11!&)!'/>!I"_U"M8!%'4!.%*,1!.&3&+&!9&//(/'+!4&6&9).!-1!7/'1).!-16&9)&4!,-)%!'!7$OK"!

7/'.;-4!9*1)'-1-1:!'!.&R(&19&!4&.-:1&4!)*!.-/&19&!Q)<OYO<U!I)%&!*+:'1&//'+!OP'.&!<!-1!

84#0(9/-)()M>!?*,&3&+8!1*! 9/&'+! 9&//(/'+!7%&1*)07&!9*(/4!5&!*5.&+3&4! -1!7/'1).!,-)%!'!

KL\S!':'-1.)!Q)<OYO<">!Q99*+4-1:!)*!*(+!+&.(/)8!)%-.!-.!3&+0!/-2&/0!4(&!)*!'!4-66&+&19&!-1!

)%&!7'+)!*6!)%&!7/'1)!.';7/&4!)*!7&+6*+;!$ZG!*5.&+3')-*1.>!

L14&&48!,%&1!.';7/-1:!)%&!;*.)!'66&9)&4!7'+)!*6!)%&!/&'68!'!9/&'+!7%&1*)07&!9'1!5&!.&&1!')!

)%&!9&//(/'+!/&3&/8!%-:%/-:%)-1:!)%&!-;7*+)'19&!*6!1(9/&'+!OP'.&!<>!?*,&3&+8!-1!7'+).!*6!)%&!

/&'3&.!)%')!4*!1*)!&B%-5-)!'!.&3&+&!;'9+*.9*7-9!7%&1*)07&8!)%&!9&//(/'+!4&6&9).!'+&!*1/0!

;-/4! IA-:(+&! #UM8! -14-9')-1:! )%')! -1! )%&.&! /&'3&.8! )%&! /&3&/! *6! 1(9/&'+! OP'.&! <8! '/5&-)!

4&9+&'.&4!-.!.)-//!.(66-9-&1)!6*+!9&//.!)*!6(19)-*1!7+*7&+/0>!

!

Q/)*:&)%&+8!)%&!O$JR<HO!4')'!'14!)%&!9&//(/'+!*5.&+3')-*1!*6!7/'1).!4*,1J+&:(/')&4!6*+!

1(9/&'+!OP'.&!<!.(::&.)!)%')!Q)GQAU!-.!-14&&4!*3&+J&B7+&..&4!')!)%&!)+'1.9+-7)*;-9!/&3&/!

,%&1! 1(9/&'+! OP'.&! <! -.! 4*,1J+&:(/')&4>! ?*,&3&+8! 4*,1J+&:(/')-*1! *6! 84#0(9/-)()!

1(9/&'+! OP'.&! <! /&'4.! )*! '1! -;7*+)'1)! .)+&..! ')! )%&! 9&//(/'+! /&3&/! '14! 6*+! )%&! &1)-+&!

*+:'1-.;>!$%(.8!.-19&!GQAU!-.!21*,1!)*!5&!'!.)+&..!+&:(/')&4!7+*)&-18!')!)%-.!.)':&8!,&!

9*(/4!1*)!+(/&!*()!)%')!)%&!-19+&'.&!-1!Q)GQAU!;OPQ!/&3&/!-.!.*/&/0!4(&!)*!'!/*,&+!';*(1)!

*6! Q)<OYO<"d#8! 5()!;-:%)! '/.*! 5&! '! +&.7*1.&! )*! )%&! .)+&..! 9'(.&4! 50! )%-.! 4&7/&)-*1>!

H*1.&R(&1)/08!-1!*+4&+!)*!)&.)!)%&!4-+&9)!&66&9)!*6!Q)<OYO<"d#!'9)-3-)0!*1!Q)GQAU!$DS8!

we used 3’RACE to test whether AtMAF1 TLS is indeed cleaved ($*7(7/!')!-).!.)'+)!.-)&>!

!

.? 3’ RACE reveals accumulation of MAF1 transcripts

termini at the 5’ extremity of the TLS*!Before selecting 3’ RACEJS&R!)*!'1'/0.&!Q)GQAU!)+'1.9+-7)!&14.8!,&!-1-)-'//0!7&+6*+;&4!

5’ RACE. In a pilot assay we had used Sanger sequencing of multiple 5'9)&+-'/!9*/*1-&.!

containing an insert with the 5’ RACE final PCR products. We could not conclude from

)%&.&!6-+.)!+&.(/).8!'.!,&!4-4!1*)!21*,!-6!)%&!;*+&!)%'1!*1&!%(14+&4!.&R(&19&.!'1'/0.&4!

9*++&.7*14&4! *+! 1*)! )*! <HO! 4(7/-9')&.>! L1! '44-)-*1! S'1:&+! .&R(&19-1:! 4-4! 1*)! 0-&/4!

&1*(:%!4&7)%!)*!9*++&9)/0!'..&..!Q)GQAU!;OPQ!)+'1.9+-7)!&14.>!$%&!;'-1!7+*5/&;!/-&.!

! UU"!

-1! )%')! )%&!$DS! -.! 6*(14! -1! '1! -1)+*1! *6!GQAU! '14! -1! 9*1.&R(&19&! *1/0! *1! 7+&J;OPQ!

.&R(&19&.!)%')!'+&!1*)!'5(14'1)!9*;7'+&!)*!)%&!;')(+&!;OPQ>!

$%(.!-1!*+4&+!)*!4&)&+;-1&!-6!Q)GQAU!$DS!-.!7+*9&..&4! ($*7(7/!'14!;-:%)!)%(.!+&:(/')&!

AtMAF1 transcriptional level, we decided to analyse the 3’ ends of the AtMAF1 transcripts

using 3’ rapid amplification of complementary ends followed by NGS sequencing (3’RACEJ

S&RM>! $*)'/! OPQ! ,'.! &B)+'9)&4! 6+*;!]$! 7/'1).8! 6+*;! 7/'1).! 21*92&4! *()! 6*+! -4/4-^!

IQ$T\"Ub__M8!'14!'6)&+!-16&9)-*1!&-)%&+!50!'!KL\S!9*1.)+(9)!)*!4*,1J+&:(/')&!Q)<OYO<"!

IQ$"\U[[^_M! *+! ,-)%! '1! &;7)0! 3&9)*+! IZKM>! A-1'//08! '! 3.$TNT! ;()'1)! I$J=PQ! /-1&!

\`oggT?_g!IPTgT"bbM!9%'+'9)&+-.&4!50!D'1:&!&)!'/>8!"_UUM!,'.!'/.*!(.&4>!$%&!'9)-3-)0!*6!

)%&!1(9/&'+!&B*.*;&!)%')!;&4-')&.!3’J5’ RNA degradation activity is reduced in this line>!

]&!&B7&9)!)%')!4&:+'4')-*1!507+*4(9).!*6!<OYO<!9/&'3':&!*6!GQAU!$DS!'+&!4&9+&'.&4!-1!

)%-.!/-1&>!L1!9*1.&R(&19&8!-)!.%*(/4! -;7+*3&!4&)&9)-*1!of the true 3’ end of the cleavage

7+*4(9)! *6! <OYO<>! In the 3’ RACE assays we used a 3’ adaptor containing a random

.&R(&19&!*6!U^!1(9/&*)-4&.!'//*,-1:!)*!+&;*3&!<HO!4(7/-9')&.!6+*;!)%&!'1'/0.-.!'6)&+!P\S!

.&R(&19-1:!*6!)%&!6-1'/!<HO!7+*4(9).>!

Results from the 3’ RACEJS&R!'+&!.%*,&4!'.!'!9/*.&J(7!6*+!)%&!-1)+*1![!I6+*;!7*.-)-*1!UU8!

6*+,'+4!7+-;&+!/*9')-*18!)*!#U[8!)%&!&14!*6!)%&!-1)+*1![M!5()!7&+9&1)':&.!'+&!9'/9(/')&4!

6+*;! )%&! )*)'/! *6! 1(;5&+! *6! +&'4.! 6*(14! '/*1:! )%&! #_[! 1(9/&*)-4&.! /*1:! ';7/-6-&4!

.&quence analysed by 3’RACEJS&R!IA-:(+&!#"Q8!H8!=M>!$%&!$DS!-.!/*9'/-.&4!')!7*.-)-*1!U#^J

"_b! -1!*(+! .1'7J.%*)! I5/(&!5'92:+*(14! -1!A-:(+&!fM>!]&!9'1! 3-.('/-@&!7&'2.! 6+*;! )%&!

7*.-)-*1.!JU!)*!J#!6+*;!)%&!.)'+)!*6!)%&!$DS!I7*.-)-*1!U#T8!U##8!U#"M!-1!)%&!]$8!-4/4-^*I7#!

`YM8!-4/4-^J3-:.ZK!I7#!`Y!c!3-:.!ZKM!'14!3.$"!5'92:+*(14.!IA-:(+&!#"M>!$%&!9/*.&J(7!

3-&w around the TLS 5’ extremity (F-:(+&!#"C8!H8!=M8!.%*,.!)%')!)%&!.(;.!*6!)%&.&!7&'2.!

+&7+&.&1)! '+*(14! T_n! *6! )*)'/! +&'4.! -1!]$! IA-:(+&! #"Q8! CM8! T"n! -1! 7#! `Y8! [fn! -1!

7#!`Y!c!3-:.!ZK! IA-:(+&!#"HM!'14!gUn! -1!3.$"!5'92:+*(14.! IA-:(+&!#"=M>!?&19&8! -)! -.!

9/&'+!)%')!'1!&14*+-5*1(9/&*/0)-9!9/&'3':&!*99(+.!'+*(14!)%&!.)')&4!7*.-)-*1.>!?*,&3&+8!

)%&!3’end byJ7+*4(9).!+&.(/)-1:!6+*;!)%&!9/&'3':&!'+&!1*)!')!'!7+&9-.&!7*.-)-*1!3'+0-1:!

5&),&&1! J#! )*! J1 in our results. The 3’ extremity in 3.$"!5'92:+*(14!,%&+&! &B*.*;&!

activity on the 3’ end should be abolished is also not clear between these positions while

)%&! 7&+9&1)':&! *6! +&'4.! -.! )%&! %-:hest compare to other backgrounds. The blurred 3’

&B)+&;-)0! 9*(/4! 5&! )%&! +&.(/)! *6! )%&! 1-55/-1:! 50! &B*+-5*1(9/&'.&.! '6)&+! )%&!

&14*+-5*1(9/&*/0)-9! 9/&'3':&! *+! '1! -;7+&9-.&! 9/&'3':&! 50! )%&! &14*+-5*1(9/&'.&!

! UU#!

+&.7*1.-5/&!*6! )%-.! 9()>! L!,*(/4! /-2&! )*! .)+&..! )%')!<OYO<!&1@0;&.!9'1!%'3&! -;7+&9-.&!

9/&'3':&! /&'4-1:! 6*+! &B';7/&! )*! )%&! &B)+'! 1(9/&*)-4&! 6*(14! ')! 7*.-)-*1! JU! -1! )OPQ!?-.!

I</'9-4*!&)!'/>!"_U_M>!$%&!4*,1+&:(/')-*1!*6!<OYO<"!-1!-4/4-^!I7#!`Y!c!3-:.!<"!-1!A-:(+&!

#"Q8!CM!.&&;.!)*!+&.(/)!-1!'!+&4(9)-*1!*6!)%&!7&+9&1)':&!*6!)+'1.9+-7)!&14.!')!7*.-)-*1!U#"8!

U##!'14!U#T>!?*,&3&+8! )%&! )%-+4!+&7/-9')&!5&%'3&!1*)! /-2&! )%&! ),*! 6-+.)!*1&!'14!&B)+'!

+&7/-9')&.!'+&!1&&4&4!)*!9*16-+;!)%&!+&.(/)>!L1!'10!9'.&8!)%&!)%+&&!7&'2.!+&7+&.&1)!*1/0!"^!

n!*6!)*)'/!+&'4.!-1!)%&!),*!6-+.)!+&7/-9')&.!5()!^_!n!-1!)%&!/'.)!+&7/-9')&>!L6!,&!)'2&!-1)*!

'99*(1)! *1/0! )%&! ),*! 6-+.)! +&7/-9')&.8! -)! -.! '! #gn! 4&9+&'.&! 9*;7'+&! )*! )%&!]$8! ^"n!

9*;7'+&!)*!7#!`Y!'14![#n!9*;7'+&!)*!7#!`Y!c!3-:.!ZK>!$%-.!+&.(/)!.&&;.!)*!.(77*+)!*(+!

%07*)%&.-.!)%')!Q)<OYO<"d#!'+&!-14&&4!+&.7*1.-5/&!6*+!)%&!9/&'3':&!*6!Q)GQAU!$DS! ($*

7(7/>!A(+)%&+!+&7/-9')&.!'+&!1&&4&4!6*+!'//!)%&!:&1*)07&.!)*!.)+&1:)%&1!)%&!+&.(/)>!!

$%&!'1'/0.-.!*6!3’ &14.!50J7+*4(9).!'//!'/*1:!)%&!.)(4-&4!.&R(&19&!I-1)+*1![!–!&B*1!gM!

.%*,.!)%')!7*/0JQ!)'-/!IQJ+-9%!c!*1/0QM!-.!)%&!;'e*+!'44-)-*1!I-1!+&48!A-:(+&!#"!'14!A-:(+&!

##QM>!]%&1!)%&!&B*.*;&!'9)-3-)0!-.!+&4(9&4!I3.$"!5'92:+*(14M8!)%&!7&+9&1)':&!*6!7*/0JQ!

-19+&'.&.!'5*3&!g_n!5&9'(.&!)%&.&!7*/0JQ!)'-/&4!50J7+*4(9).!'+&!'99(;(/')-1:!IA-:(+&!

##QM>!$%&.&!'+&!,&//!21*,1!.(5.)+')&.!*6!)%&!&B*.*;&!ID'1:&!&)!'/>8!"__bM>!$%&!6*9(.!*1!

;*4-6-9')-*1.!')!7*.-)-*1.!U#"8!U##8!U#T!5&6*+&!)%&!$DS!IA-:(+&!##CM!9/&'+/0!.%*,.!7*/0J

Q!)'-/!'.!)%&!;'-1!;*4-6-9')-*1>!$%&!9/&'3':&!50J7+*4(9).!'+&!)%&+&6*+&!)%&!)'+:&)!*6!)%&!

&B*.*;&>!S-19&!)%&!'44-)-*1!*6!7*/0JQ!)'-/.!,'.!4&.9+-5&4!'.!'!.-:1'/!6*+!OPQ!4&:+'4')-*1!

ID'1:&!&)!'/>8!"__bM8!)%-.!.)+&1:)%&1.!)%&!-4&'!)%')!)%&.&!)+'1.9+-7).!'+&!4&:+'4&4!50!)%&!

OPQ!4&9'0!;'9%-1&+0! '6)&+! &14*+-5*1(9/&*/0)-9! 9/&'3':&>! ! Y3&+'//8! )%&! +&.(/).! .(::&.)!

)%')!1(9/&'+!<OYO<.!'+&!+&.7*1.-5/&!6*+!)%&!&14*+-5*1(9/&*/0)-9!9/&'3':&!*6!GQAU!7+&J

mRNA at the 5’ end of a TLS in intron 6. Upon cleavage, the byJ7+*4(9)!/*9')&4!(7.)+&';!

)%&!9/&'3':&!.-)&!-.!4&:+'4&4!50!)%&!1(9/&'+!&B*.*;&!'..*9-')&4!,-)%!)%&!%&/-9'.&!?ZP">!

]&!9*(/4!1*)&!)%')!)%&!9/&'3':&!*99(+.!-1!)%&!]$!-1!.)'14'+4!:+*,)%!9*14-)-*1.>!

! UUT!

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0

10

20

30

10

20

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10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

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rep3

P2

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020406080002040608000204060800020

10

20

30

10

20

30

10

20

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X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

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onlyGrep3

WT

rep3

P2

rep2

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rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

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10

20

30

10

20

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10

20

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X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyGrep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

0

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

10

20

30

0

10

20

30

10

20

30

X3.end

percent

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

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rep3

P2

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rep1

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rep1

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hen2

020406080002040608000204060800020

10

20

30

10

20

30

0

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

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onlyC

ich

onlyG

!

"

rep3

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rep3

P2

rep2

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rep2

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hen2

rep1

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rep1

P2

rep1

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10

20

30

0

10

20

30

10

20

30

X3.end

percent

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

0 2 4 6 8 0 2 4 6 8 0

10

20

30

10

20

30

0

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

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rep2

P2

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hen2

rep1

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rep1

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rep1

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10

20

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10

20

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10

20

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X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

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onlyC

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onlyG

rep3

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rep3

P2

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rep2

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hen2

rep1

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rep1

P2

rep1

hen2

10

20

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10

20

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10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

20 22 24 26 28 30 32 34 36 38 40

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

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#"

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10

20

30

10

20

30

10

20

30

X3.end

class_ord

No−mod

A−rich

onlyA

U−rich

onlyU

C−rich

onlyC

G−rich

onlyG

## $#%#$& '()*+,-./01.2%

## $#%#$& '()*+,-./01.2%

#$&

*+,-./01.2%

#'# #$&

*+,-./01.2%

#'#

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

020406080002040608000204060800020

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

!"

#"

$"

"

!"

#"

$"

"

!"##

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

rep3

WT

rep3

P2

rep2

WT

rep2

P2

rep2

hen2

rep1

WT

rep1

P2

rep1

hen2

20 22 24 26 28 30 32 34 36 38 40

10

20

30

10

20

30

10

20

30

X3.end

class_ord

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

onlyG

!"##

rep2

EV

rep2

P3KO

0 20 40 60 80 00 20 40 60 80 00 20 40 60 80 00 20

10

20

30

40

X3.end

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

="

!"

#"

$"

"

%!&'(3

4

="

!"

#"

$"

"

rep2

EV

rep2

P3KO

20 22 24 26 28 30 32 34 36 38 40

10

20

30

40

X3.end

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

%!&'(

#$&

*+,-./01.2%

#'# rep2

EV

rep2

P3K

20 22 24 26 28 30 32 34 36 38 40

0

10

20

30

40

X3.end

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

%!&'(&)&*+,- >?

#$&

*+,-./01.2%

#'#

## $#%#$& '()*+,-./01.2%

rep2

EV

rep2

P3K

0 20 40 60 80 00 20 40 60 80 00 20 40 60 80 00 20 0

0

10

20

30

40

X3.end

No mod

ich

onlyA

ich

onlyU

ich

onlyC

ich

%!&'(&)&*+,- >?

## $#%#$& '()*+,-./01.2%

## $#%#$& '()

*+,-./01.2%

#$&

*+,-./01.2%#'#

.123145&67&56589&218:-

.123145&67&56589&218:-

U!

"!

#!

U!

"!

#!

U!

"!

#!

U!

"!

#!

U!

"!

U!

"!

! UU^!

F2*L0(.64N!Analysis of the 3’RACEA-(N.0(-L',-.-R%&2$*."'(313*(./DAproducts 3’ ends along the intron

[!IQ8!'14!/&6)!7'1&/.!*6!H!'14!=M!'14!6*+!'!9/*.&Jup around the 5’ end of the TLS (B, right panels of C and D)

6*+! )%&! 4-66&+&1)! :&1*)07&.>! O&.(/).! '+&! &B7+&..&4! '.! 7&+9&1)':&! *6! )*)'/! +&'4.! 6*+! )%&!,%*/&! .&R(&19&!

'1'/0.&4! -1! )%-.! &B7&+-;&1)>! $%&! 9*/*(+.! &B7+&..! )%&! +&'4.! (1;*4-6-&4! I5/'92M8! +&'4.! ,-)%! 7*/0JQ!

(1)&;7/')&4!'44&4!1(9/&*)-4&.!IQJ+-9%!*+!*1/0!Q!-1!+&4!9*/*(+.M!'14!+&'4.!,-)%!*)%&+!)07&.!*6!(1)&;7/')&4!

'44&4!1(9/&*)-4&.!I-1!:+&0M>!$%+&&!+&7/-9')&.!*6!)%&!&B7&+-;&1)!6*+!]$!'14!7#!`Y!c!3-:.!<"!'+&!7+&.&1)&4!

-1!Q!'14!C8!*1&!&B7&+-;&1)!,'.!4*1&!6*+!7#!`Y!'14!7#!`Y!c!3-:.!ZK!IHM!'14!),*!+&7/-9')&.!,&+&!4*1&!6*+!

3.$"!I=M>!

!

F2*L0(. 66N! !))2,2%$. %W. (d,03A$L"'(%,2)(-. 3,. ,R(. ($). %W. "'(313*(. /DAP0%)L",-! '/*1:! )%&! '1'/0.&4!

.&R(&19&!IQM!'14!')!7*.-)-*1!U#"8U##8!U#T!9*++&.7*14-1:!)*!JU!)*!J3 upstream the 5’end of the TLS (B) for

)%&!4-66&+&1)!:&1*)07&.!)&.)&4>!$%&!9*/*(+!9*4&!-.!4&.9+-5&4!*1!)%&!+-:%)>!

! UU[!

!!

F2*L0(. 6YN!G03PR2"3'. 0(P0(-($,3,2%$. %W. ;!8EA-(N. )3,3. W0%+. !03/2)%P-2-. @(d,AG($. )3,3/3-(. W%0.

!,:!FT.*($(!IQ)^:U#"T_M>!$%&!7*.-)-*1!*6!)%&!$DS8!-1!GQAU!-1)+*1![!-.!-14-9')&4!50!'1!'++*,!IQM>!$DS!

.&R(&19&!-.!-14-9')&4!-1!+&48!'14!)%&!.&R(&19&!*6!)%&!+&'4!6*(14!-1!)%&!P&B)J\&1!4')'5'.&!-.!-14-9')&4!5&/*,!

-1!:+&&1>!$%&!.)'+)!.-)&!*6!)%&!$DS!-.!-14-9')&4!50!'1!'++*,!ICM>!

!

! !

! UUg!

C&0*14! *(+! *,1! &B7&+-;&1)'/! 3’OQHZJS&R! +&.(/).8! ,&! '/.*! (.&4! '3'-/'5/&! <QOZJ.&R!

I7'+'//&/! '1'/0.-.! *6! OPQ! &14.M! 4')'! produced by the Pamela Green’s lab (publicly

'3'-/'5/&!')!1&B)J:&1!.&R(&19&M>!<QOZJ.&R!-.!'!)&9%1-R(&!(.&4!)*!.)(40!OPQ!4&:+'4')-*1!

'14!-4&1)-60!6*+!&B';7/&!;-OPQ!9/&'3':&!.-)&.>!!]%&1!6*9(.-1:!*1!GQAU8!)%&!1(;5&+!*6!

)+'1.9+-7)!&14.!.)'+).!)*!5&!'5*3&!5'92:+*(14!')!)%&!&xact 5’ start site of the TLS (F-:(+&!

#TM!'14!)%-.! -.! )%&!*1/0!7&'2! /*9'/-.&4! -1!'1! -1)+*1>! L!,*(/4! /-2&!)*!.)+&..!)%')! )%&!$DS!

.&R(&19&!-.!4-66&+&1)!&1*(:%!6+*;!)%&!)OPQ!.&R(&19&.!6*+!)%&!+&'4.!)*!5&!.7&9-6-9>!$%-.!-.!

'1! '44-)-*1'/! 7+**6! )%')! '! &14*+-5*1(9/&*/0)-9! 9/&'3':&! +&'//0! *99(+.! -1! )%-.! -1)+*1! [!

&B'9)/0!at the 5’ end of the transcript.!

!

B? 8$#:D)()*/B*@;8*-/:D&.4#).*666*%4#$)<4(-%)*>3.$*2@K@2Tf^*()*9/>$4.A":#%.9*

!Q.! '! 1&B)! .)&78! '6)&+! .%*,-1:! )%')! Q)GQAU! ;OPQ! /&3&/! ,'.! /-2&/0! '/.*! +&:(/')&4! 50!

Q+'5-4*7.-.!1(9/&'+!OP'.&!<!,&!4&9-4&4!)*!-13&.)-:')&!-68!-1!*(+!KL\S!9*14-)-*1.!,%&+&!

Q)<OYO<#! -.! '5.&1)! '14! Q)<OYO<"! -.! 4*,1J+&:(/')&48! OPQ! )+'1.9+-7).! 6+*;! OPQ!

7*/0;&+'.&! LLL! '+&! '/.*! 4*,1J+&:(/')&4>! L14&&48! -1! *(+! 7+*7*.&4!;*4&/8! Q)<OYO<"d#!

9')'/0.&!)%&!9/&'3':&!*6!Q)GQAU!7+&9(+.*+!;OPQ8!)%&+&50!+&4(9-1:!)%&!/&3&/!*6!-).!;')(+&!

;OPQ!'14!9*1.&R(&1)/0!7*..-5/0!-).!'5(14'19&!')!)%&!7+*)&-1!/&3&/!'14!-).!+&7+&..-*1!*6!

OPQ!7*/! LLL>!Q99*+4-1:/08!,&!%07*)%&.-.&4! )%')! )%&!4*,1+&:(/')-*1!*6!Q)<OYO<"d#!50!

KL\S!;-:%)! /&'4! )*!'1!*3&+'//!4&9+&'.&!*6! OPQ!7*/! LLL! )+'1.9+-7).>! L)!%'4!'/+&'40!5&&1!

.%*,1!50!7+&3-*(.!,*+2!6+*;!)%&!)&';!)%')!,%&1!Q)<OYO<"d#!'+&!4*,1+&:(/')&48!)%&!

';*(1)! *6! .*;&!;')(+&! )OPQ.! -.! '/.*! 4&9+&'.&4! I\();'11! &)! '/>8! "_U"M8! '/)%*(:%!1*!

)+'1.9+-7)*;&!,-4&!'1'/0.-.!%'4!5&&1!7&+6*+;&4>!S*!,&!4&9-4&4!)*!-13&.)-:')&!-6!)%-.!,'.!

'/.*!)%&!9'.&! 6*+!*)%&+!OPQ!7*/! LLL! )+'1.9+-7).>!E.-1:!O$JR<HO!,&!'..'0&4!)%&!+&/')-3&!

&B7+&..-*1!/&3&/!*6!)%+&&!*)%&+!OPQ!7*/!LLL!)+'1.9+-7).N!^S!+-5*.*;'/!OPQ8!gSD!OPQ!'14!

E[!.7/-9&*.*;'/!OPQ>!$%&!'1'/0.-.!*6! )%+&&! -14&7&14&1)!&B7&+-;&1).!+&3&'/&4!)%')!^S!

+OPQ8!E[!.1OPQ!'14!gSD!OPQ!/&3&/.!,&+&!1*)!.-:1-6-9'1)/0!4&9+&'.&4!-1!-4/4-^*[K!7/'1).!

,-)%!3-:.!':'-1.)!<OYO<"!.';7/&.!9*;7'+&4!)*!-4/4-^!`Y!7/'1).!,-)%!3-:.!50!'1!Z;7)0!

K&9)*+!.';7/&.!IA-:(+&!#^M>!?*,&3&+8!)%&.&!;&'.(+&.!;'0!+&3&'/!)%&!.)&'40J.)')&!/&3&/!

*6!)%&.&!)+'1.9+-7).8!'14!1*)!4-+&9)/0!OPQ!7*/!LLL!'9)-3-)0>!!

!

! UUf!

!

F2*L0(. 6ZX. 8('3,21(. 8@!. P%'. BBB. ,03$-"02P,-. '(1('-. 2$. P'3$,-.&2,R. >BG9. 3*32$-,. ;8J8;4! -1! -4/4-^!

5'92:+*(14! 9*;7'+&4! )*! 7/'1).! ,-)%! KL\S! 50! '1! &;7)0! 3&9)*+! IZKM! '..'0&4! 50! O$JR<HO>! S)'14'+4!

4&3-')-*1.!'+&!-14-9')&4!50!7*.-)-3&!&++*+!5'+.>!O&/')-3&!6*/4!9%'1:&!&B7+&..-*1!/&3&/!9*;7'+&4!)*!9*1)+*/8!

-14-9')&4!*1!)%&!V!'B-.8!,'.!9'/9(/')&4!(.-1:!)%&!4&/)'J4&/)'!H)!;&)%*4>!=-66&+&19&!-1!/&3&/!*6!&B7+&..-*1!

5&),&&1!)%&!),*!9*14-)-*1.!IKL\S!':'-1.)!<OYO<"!9*;7'+&4!)*!KL\S!':'-1.)!&;7)0!3&9)*+M!,'.!'..'0&4!

using student’s tJ)&.)!'14!9*1.-4&+&4!.)')-.)-9'//0!.-:1-6-9'1)!-6!7!z!_8_^!I-14-9')&4!50!'!.)'+M>!Y)%&+,-.&8!

4-66&+&19&!,'.!9*1.-4&+&4!1*)!.)')-.)-9'//0!.-:1-6-9'1)!I1>.M>!

!

! !

! UUb!

A? 8)).))&.$%*/B*#*-/))(0:.*B"$<%(/$*/B*@;#).*b*B/4*%3.*<:.#7#A.*/B*H8=I*GR'**

!As presented in the introduction, precursor tRNAs are matured in 5’ by RNase P and in 3’

50!OP'.&!W>!]%-/&!,&!%'4!-4&1)-6-&4!)%')!OP'.&!<!9/&'3&.!)%&!9*1.&+3&4!Q)GQAU!$DS!-1!

*+4&+!)*!+&:(/')&!Q)GQAU!/&3&/.8!-)!,'.!'/.*!-;':-1'5/&!)%')!OP'.&!W!,*(/4!%'3&!'!.-;-/'+!

regulatory function through a cleavage of the 3’ extremity of AtMAF1 TLS. We therefore

4&9-4&4!)*!'44+&..!)%-.!%07*)%&.-.!5*)%!($*7(%4/!'14!($*7(7/>!

Q.!'!6-+.)!.)&78!,&!-13&.)-:')&4!-6!Q)GQAU!$DS!9*(/4!5&!9/&'3&4!50!'!+&9*;5-1'1)!Q)OP'.&!

W#!7+*)&-1!I)%&!1(9/&'+!Q+'5-4*7.-.!OP'.&!W!'99*+4-1:!)*!H'1-1*!&)!'/>8!"__bM! ($*7(%4/>!

O&9*;5-1'1)!Q)OP'.&!W#!,'.!7+*4(9&48!6*//*,-1:!)%&!7+*)*9*/!4&)'-/&4!-1!)%&!G')&+-'/!

'14!;&)%*4.!.&9)-*18!,-)%!)%&!H=S!6*+!Q)OP'.&!W#!9/*1&4!-1)*!7&)"f5!'14!&B7+&..&4!6+*;!

O*.&))'!=Z#!9&//.>!Q)OP'.&!W#!9*(/4!5&!7(+-6-&4!)*!1&'+!%*;*:&1&-)0!'.!3&+-6-&4!*1!:&/!

'14!50! -;;(1*4&)&9)-*1! IA-:(+&!#[M8! '14!50!;'..! .7&9)+*;&)+0!'1'/0.-.>!$%&! .';7/&!

6+*;!$OW#!&/()-*1!U!,'.!(.&4!6*+!'9)-3-)0!'..'0.>!

H/&'3':&! '..'0.! ,&+&! 7&+6*+;&4! '.! 4&.9+-5&4! '5*3&8! ,-)%! )%&! .%*+)! )+'1.9+-7)!

+&7+&.&1)-1:!GQAU!$DS!'14!'!7+&J)OPQ!'.!'!7*.-)-3&!9*1)+*/>!$+'1.9+-7).!,&+&!-19(5')&4!

,-)%!&-)%&+!Q)OP'.&!W#!'/*1&8!Q)<OYO<"!'/*1&!*+!,-)%!5*)%!7+*)&-1.>!O&.(/)!.%*,&4!)%')!

Q)GQAU!$DS!9*(/4! -14&&4!5&!9/&'3&4!50!Q)OP'.&!W#!5()!;(9%!;*+&!&66&9)-3&/0! -1!)%&!

7+&.&19&!*6!Q)<OYO<"!IA-:(+&!#gM>!Q!.-;-/'+!+&.(/)!,'.!*5.&+3&4!,-)%!)%&!9*1)+*/!)OPQ>!

$%-.!.(::&.).!)%')!OP'.&!<!'9)-3-)0!;(.)!7+&9&4&!OP'.&!W!9/&'3':&!'14!)%')!only 5’ mature

)OPQ.!*+!$DS!'+&!:**4!.(5.)+')&.!6*+!OP'.&!W>!P(9/&'+!7+&9(+.*+!)OPQ.!'+&!5*(14!($*7(7/!

by the LA protein at their 3’ extremity. This protein was proposed to prevent RNase Z

&14*1(9/&*/0)-9!9/&'3':&!'14!)%(.!)*!*+-&1)!7+&J)OPQ.!)*,'+4.!;')(+ation of their 5’ end

6-+.)!IC/&,&))!'14!G'+'-'8!"_UfM>!Y(+!+&.(/).!*5)'-1&4!($*7(%4/!.(::&.)!)%')!)%&!DQ!7+*)&-1!

-.!1*)!)%&!;'-1!7/'0&+!)*!4+-3&!)%&!7+&6&+&1)-'/!9/&'3':&!50!OP'.&!<!6-+.)>!$%&!1')(+&!*6!

)%&!7+&J)OPQ!.(5.)+')&! -).&/6! .&&;.! )*!5&! .(66-9-&1)! )*! -14(9&!7+&6&+&1)-'/! 9/&'3':&! 50!

OP'.&!<!6-+.)8!-1!'99*+4'19&!,-)%!7+&3-*(.!/-)&+')(+&!I?*77&+8!"_U#M>!

!

!

! U"_!

!

F2*L0(.6[X.V(-,(0$./'%,.3$3'D-2-.%W.!,<8Q6AHB9!I9'/9(/')&4!G]N!bf8[!`4'M!'6)&+!7+*)&-1!&B7+&..-*1!-1!

5'9)&+-'/!9&//.!'14!5')9%!7(+-6-9')-*1>!]'.%!I]M8!Z/()-*1!IZDM!'14!A/*,!$%+*(:%!IA$M!.';7/&.!,&+&!+(1!*1!

'1!fn!S=SJ<Q\Z!:&/>!Q6)&+!)+'1.6&+!)*!'!<K=A!;&;5+'1&8!,&.)&+1!5/*))-1:!,'.!7&+6*+;&4!(.-1:!'1)-5*40!

':'-1.)!?LS>!G-:+')-*1!7*.-)-*1.!*6!)%&!;*/&9(/'+!;'..!;'+2&+.!'+&!-14-9')&4!*1!)%&!/&6)!IGM>!!

!

F2*L0(.6\X.B$.12,0%."'(313*(.3--3D.%W.:!FT.<K9./D.!,;8J8;4.3$).!,8@3-(.Q6O!Q!.%*+)!+&:-*1!9'//&4!S!

OPQ!I9*++&.7*14-1:!)*!'!7*+)-*1!'+*(14!)%&!$DS!*6!-1)+*1![!*6!GQAUM!,'.!)+'1.9+-5&4!($*7(%4/!'14!(.&4!'.!

'!.(5.)+')&!6*+!+&9*;5-1'1)!<OYO<"!I<"M!'14d*+!OP'.&!W#!IW#M>!Q.!'!1&:')-3&!9*1)+*/8!)%&!+&'9)-*1!,'.!

7&+6*+;&4! ,-)%*()! 7+*)&-1! IJM>! Q.! '! 7*.-)-3&! 9*1)+*/8! '! 7+&9(+.*+! )OPQ! I)%&! 1(9/&'+! 7+&9(+.*+! )OPQ!

H0.)&-1&M!,'.!(.&4!'.!'!.(5.)+')&8!.%*,-1:!)%')!)%&!+&9*;5-1'1)!<OYO<"!'14!OP'.&!W#!'+&!9')'/0)-9'//0!

'9)-3&>! $%&! +&'9)-*1! 7+*4(9).! ,&+&! +(1! *1! '1! '9+0/';-4&! :&/! '14! 3-.('/-@&4! (14&+! EK! '6)&+! &)%-4-(;!

5+*;-4&!.)'-1-1:>!

$OW#!J!?LS!

! U"U!

$%&18!'.!'!1&B)!.)&78!,&!-13&.)-:')&4!,%&)%&+!.(9%!'1!OP'.&!W!9/&'3':&!9*(/4!*99(+!')!)%&!

/&3&/!*6!GQAU!$DS!($*7(7/. For this, we analysed our 3’ RACE Seq assays. This data did not

+&3&'/!'10!'99(;(/')-*1!*6!GQAU!)+'1.cripts at the specific site at the 3’ end of the TLS!

I7*.-)-*1!"_bM!5()!,&!9'1!*5.&+3&!'!.;'//!-19+&'.&!-1!7&+9&1)':&!*6!+&'4.!')!7*.-)-*1!"UU!

*1/0!6*+!7#!`Y!3-:.!<"!+&7/-9')&!"!IA-:(+&!#"QM>!

?*,&3&+8!-6!)%&!+&.(/).!6+*;!*(+!7+&3-*(.!($*7(%4/!'..'0.!IA-:(+&!#gM!'+&!9*++&9)!'14!)%')!

($*7(7/8!OP'.&!W!9/&'3':&!'/.*!;'-1/0!6*//*,.!OP'.&!<!9/&'3':&8!-)!-.!/*:-9'/!)%')!,&!9'11*)!

detect any RNase Z cleavage using 3’ RACE. Indeed, the adapter would then ligate itsel6!-1!

3’ of the TLS, but we would not be able t*!';7/-60!)%-.!6+':;&1)!'.!*(+!A*+,'+4!7+-;&+.!

are located in 5’ of the TLS. Consequently, once an RNase P cleavage has happened, if

another cleavage occurs, catalysed by RNase Z, we cannot amplify by PCR until the 3’end

*6!)%&!$DS8!'.!)%&1!)%&!OPQ!-.!'/+&ady cleaved. Consequently, with 3’RACE we are only

'5/&!)*!4&)&9)!OP'.&!W!9/&'3':&!-6!-)!*99(+.!5&6*+&!OP'.&!<!9/&'3':&>!Q14!,&!%'3&!.%*,1!

($* 7(%4/! )%')! )%-.! -.!(1/-2&/0! )%&! 9'.&>!H*1.&R(&1)/08! -6!,&!,'1)! )*! -13&.)-:')&! ($* 7(7/! -6!

Q)GQAU!$DS!-.!9/&'3&4!50!RNase Z, it is necessary to perform a 5’ RACE assay.!!D-2&,-.&8!

,&!'/.*!-1)&++*:')&4!7(5/-9!4')'5'.&.>!A*+!-1.)'19&8!)%&!'1'/0.-.!*6!)%&!19OPQ!9*//&9)&4!

'14! ! '..&;5/&4! *1! )%&! Q+'5-4*7.-.! P&B)J\&1! .&R(&19&! =')'C'.&.! 6*+! )%&! Q)GQAU!

IQ)^:U#"T_M!.%*,&4!)%')!1*!.;'//!OPQ8!->&>!)%')!,*(/4!9*++&.7*14!)*!GQAU!$DS8!'77&'+.!

)*!5&!:&1&+')&4!6+*;!)%-.!:&1&>!A(+)%&+;*+&8!Q22(+')*3!&)!'/>!I"_UTM!%'4!'/.*!.&'+9%&4!

6*+!'!1*1J9*4-1:!OPQ!9*1.-.)-1:!*6!)%&!)OPQ!/-2&!+&:-*1!*6!GQAU!'14!9*(/4!1*)!4&)&9)!.(9%!

'1!OPQ!(.-1:!OPQ!5/*)!%05+-4-@')-*1.>! L1! 9*19/(.-*18!,&!4-4!1*)! 6-14!'10!&3-4&19&! )*!

.(77*+)!)%&!%07*)%&.-.!)%')!OP'.&!W!,*(/4!'/.*!9/&'3&!Q)GQAU!$DS8!'14!9*1.&R(&1)/0!

:&1&+')&! '! 1*1J9*4-1:! OPQ>! ?*,&3&+8! ,&! ,*(/4! 1&&4! )*! 7&+6*+;! '44-)-*1'/! '..'0.!

(especially 5’ RACE) to be a5/&!)*!9*13-19-1:/0!+&e&9)!)%-.!%07*)%&.-.>!

!

3? 6$7.)%(A#%(/$*/B*#:%.4$#%(7.*3D-/%3.)()*%/*.O-:#($*/"4*B($9($A)*

!Q1*)%&+! '/)&+1')-3&! -1)&+7+&)')-*1! *6! )%&! <QOZ! S&R! +&.(/).! 9*(/4! %'3&! 5&&1! )%')!OPQ!

7*/0;&+'.&!LL!,*(/4!.)'//!')!)%&!.7&9-6-9!$DS!.)'+)!.-)&8!/&'4-1:!)*!'5*+)&4!)+'1.9+-7)-*1!

'14!9*1.&R(&1)/0!)%&!'5(14'19&!-1!9&//.!*6!'1!Q)GQAU!7+&J;OPQ!)+'1.9+-7).!'.!*5.&+3&4!

in our 3’RACE assays. To test this %07*)%&.-.8!,&!/**2&4!')!'3'-/'5/&!H%-7JS&R!!

! U""!

!

!

!

.

F2*L0(.6]N!?RB;A-(N.P0%W2'(-.%W.8@!;BBA94;.3$).8@!;BBA9Z;.3,.,R(.!,Z*T64YC!/*9(.!(7*1!5J&.)+'4-*/!

3&+.(.!;*92!)+&');&1)>!</*).!,&+&!:&1&+')&4!,-)%!)%&!L1)&:+')-3&!\&1*;-9.!C+*,.&+!(.-1:!+&7+&.&1)')-3&!

5-*/*:-9'/!+&7/-9')&.>!L1!:&1&!;*4&/.8!)%-1!5'+.!+&7+&.&1)!-1)+*1.!'14!)%-92!5'+.!&B*1.>!<*.-)-*1!*6!-1)+*1![8!

9*1)'-1-1:!)%&!$DS!-1!Q)GQAU!-.!-14-9')&4!50!'!+&4!5*B>!Q!9/*.&J(7!3-&,!*1!Q)GQAU!IQ)^:U#"T_M!-.!7+*3-4&4!

-1!)%&!5*))*;!7'1&/>!

4')'!6*+!84#0(9/-)()!OPQ!7*/0;&+'.&!LL!IQ1)*.@!&)!'/>8!"_"_M>!Q1'/0.-.!*6!)%&-+!4')'!9/&'+/0!

.%*,.!)%')!OPQ!7*/!LL!*99(7'190!.&&;.!R(-)&!%*;*:&1*(.!,-)%-1!)%&!,%*/&!:&1&!5*40!

! U"#!

IA-:(+&! #fM>! G*+&*3&+! )%-.! +&.(/)! -.! .-;-/'+! 6*+! )%&! ),*! '1)-5*40! (.&4! I)%&.&! ),*!

'1)-5*4-&.8!1';&4!OPQ<LLJS"<!'14!OPQ<LLJS^<8!'+&!.7&9-6-9!6*+!)%&!7%*.7%*+0/')&4!6*+;.!

*6! OPQ<LLJH$=! I)%&! 9'+5*B0J)&+;-1'/! 4*;'-1! *6! )%&! /'+:&! .(5(1-)! *6! OPQ<LLMM>!

Q44-)-*1'//08!-)!-.!,*+)%!1*)-1:!)%')!)%&.&!4')'!,&+&!:&1&+')&4!50!(.-1:!'!;()')&4!3&+.-*1!

*6!)%&!&/*1:')-*1!6'9)*+!$ALLS!,%-9%!-.!+&R(-+&4!6*+!&66-9-&1)!+&.9(&!*6!'++&.)&4!OPQ!<*/>!LL!

50!.)-;(/')-1:!)%&!-1)+-1.-9!)+'1.9+-7)!9/&'3':&!'9)-3-)0!*6!)%&!7*/0;&+'.&>!H*1.&R(&1)/08!

-1!)%&-+!/-1&.8!$ALLS!'9)-3-)0!-.!5/*92&4!,-)%*()!)%&!'44-)-*1!*6!βJ&.)+'4-*/>!L1)&+&.)-1:/08!

6*+! )%&!Q)GQAU!:&1&8!OPQ!7*/>! LL!*99(7'190!4*&.!1*)! .&&;! )*!3'+0!4+';')-9'//0!(7*1!

)+&');&1)!,-)%!βJ&.)+'4-*/>! S(77*+)-1:! )%&! -4&'! )%')! )%&!*5.&+3&4!9/&'3':&! -1!Q)GQAU!

7+&9(+.*+!;OPQ!-.!1*)!/-12&4!)*!OPQ!<*/!LL!5'92)+'92-1:!d!'++&.)>!!

G*+&*3&+8! ,&! '/.*! 9*1.-4&+8! )%-.! -1)&+7+&)')-*1! '.! (1/-2&/0! 5&9'(.&! )%&! 1(;5&+! *6!

transcript termini corresponding to MAF1 TLS 5’ extremity were reduced in AtPRORP2

4*,1J+&:(/')-*1!;()'1).8! )%(.!7*-1)-1:!'!4-+&9)! +*/&!*6!OP'.&! <! 6*+! )%&!:&1&+')-*1!*6!

)%&.&!)&+;-1->!H*1.&R(&1)/08!,&!+(/&!*()!)%&!%07*)%&.-.!*+!OPQ!7*/>!LL!.)'//-1:!)*!&B7/'-1!

*(+!6-14-1:.>!

!

Q1*)%&+!7*..-5/&!&B7/'1')-*1!9*19&+1-1:!)%&!5-*/*:-9'/!6(19)-*1.!*6!Q)GQAU!$DS!-.!)%')!-)!

,*(/4!5&!-13*/3&4!-1!-1)+*1!.7/-9-1:>!Q.!)%-.!.)+(9)(+&!-.!&3*/()-*1'+0!9*1.&+3&4!-1!-1)+*1.!

'14!'.!-1)+*1.!'+&!21*,1!)*!5&!.7/-9&4!6*+!:&1&!&B7+&..-*1!)*!5&!9*++&9)/0!'9%-&3&48!-)!-.!

'!7*..-5/&!&B7/'1')-*1!)%')!Q)GQAU!$DS!,*(/4!5&!/-12&4!-1!'!,'0!)*!-1)+*1!.7/-9-1:>!$*!

-13&.)-:')&! )%-.! %07*)%&.-.8! ,&! (.&4! )+'1.:&1-9! &#BI! 2*! /-1&.! 9*;7/&;&1)&4! ,-)%!

4-66&+&1)!3&+.-*1.!*6!Q)GQAU!I)%&.&!/-1&.!'+&!4&.9+-5&4!5&/*,M>!

]&!7&+6*+;&4!O$J<HO!*1!)%&!/-1&.!%'3-1:!&-)%&+!'!3&+.-*1!*6!)%&!Q)GQAU!:&1&!,-)%*()!

)%&! $DS! *+! -1)+*1! [! *6! Q)GQAU! I)%')! 9*1)'-1.! )%&! $DSM! (14&+! )%&! 9*1)+*/! *6! -).! *,1!

7+*;*)&+!I'.!Q)GQAU!7+*;*)&+!.&R(&19&!-.!(121*,1!,&!';7/-6-&4!"!257!(7.)+&';!*6!

the 5’UTR) to see if AtMAF1 mRNA!.-@&!-.!'66&9)&4>!Y(+!+&.(/)!IA-:(+&!#bM!.%*,.!)%')!)%&!

.-@&!*6!)%&!9*4-1:!.&R(&19&!IH=SM!-.!.-;-/'+!-1!]$!/-1&.!9*;7'+&4!)*!*(+!9*;7/&;&1)&4!

/-1&.>!H*1.&R(&1)/08! -1)+*1![! -.!9*++&9)/0!+&;*3&4!&3&1! -1!)%&!'5.&19&!*6!Q)GQAU!$DS8!

-14-9')-1:!)%')!)%-.!&3*/()-*11'+0!9*1.&+3&4!$DS!-.!1*)!-13*/3&4!-1!-1)+*1!.7/-9-1:>!

! U"T!

!

F2*L0(.6^X.8<A;?8.P(0W%0+().%$.!,:!FT.?59!-1!]$!IH*/JYM!/-1&.8!&#BI!`Y!/-1&.!I&#B!\`M!'14!;'6U!`Y!

/-1&.!9*;7/&;&1)&4!,-)%!'!3&+.-*1!*6!Q)GQAU!)%')!4*&.!1*)!9*1)'-1!)%&!$DS!.&R(&19&!IJ$DSM!*+!)%&!$DS!

9*1)'-1-1:!-1)+*1!IJ-[M>!Q)GQAU!H=S!.-@&!-.![g^!5'.&!7'-+>!Y1!=PQ!)%&!&B7&9)&4!.-@&!-.!UfbU!5'.&!7'-+>!<HO!

product deposited in the line “DNA” ,'.!9*14(9)&4!*1!'!7**/!*6!=PQ!9*1)'-1:!=PQ!&B)+'9)&4!6+*;!]$8!J!

$DS!'14!–-[! /-1&.>!$%&!–-[! /-1&.!4*!1*)!%'3&!Q)GQAU!-1)+*1!1k[8!&B7/'-1-1:!)%&!7+&.&19&!*6!),*!4-.)-19)!

5'14.!4(&!)*!)%&!.-@&!4-66&+&19&>!

! !

! U"^!

(? F.7.:/-&.$%*/B*A.$.%(<*%//:)*%/*($7.)%(A#%.*#*-/))(0:.*($%.4-:#D*0.%>..$*@;#).*2*#$9*@;#).*b*#<%(7(%(.)*($*84#0(9/-)()**

!To investigate a possible role of the nuclear RNase Z in intron 6 cleavage at the 3’end of

)%&!$DS8!,&!6-+.)!:&1*)07&4!7/'1)! -1.&+)-*1!/-1&.!9%'+'9)&+-.&4!50!H'1-1*!&)!'/>! I"__bM8!

)%')! %'3&! '! $J=PQ! -1.&+)-*1! -1! Q)OP'.&WU! I\QCL! `')! [b[H_TM! '14! -1! Q)OP'.&! W#!

IHS?Do\$bf_gM8!'.!,&//!'.!'!9+*..!)%')!%'4!5&&1!;'4&!5&),&&1!)%&.&!/-1&.!50!S>!5-14&+!

IE/;! (1-3&+.-)0M>! P*! 4*(5/&! %*;*@0:*(.! 21*92! *()! 6*+! Q)OP'.&WU! '14! W#! 9*(/4! 5&!

-4&1)-6-&48!9*16-+;-1:!)%&!+&.(/).!*6!H'1-1*!&)!'/>!I"__bM>!$%&18!,&!9+*..&4!8%-4/4-^!`Y!

/-1&.!I4&.9+-5&4!50!\*5&+)!&/!'/>8!"_U_!'14!\();'11!&)!'/>8!"_U"M!,-)%!8%@;#).*L^! /-1&.!

I4&.9+-5&4! 50! H'1-1*! &)! '/>8! "__bM>! Q6)&+! %'+3&.)-1:! .&&4.! 6+*;! )%&! %*;*@0:*(.! A"!

:&1&+')-*18!,&!'1'/0.&4!7/'1).!%'3-1:!)%&!-^-^%4L^%4L^!:&1*)07&>!$%&.&!7/'1).!4-4!1*)!

&B%-5-)!'10!*53-*(.!;'9+*.9*7-9!7%&1*)07&>!$%&!+&.(/)!,'.!&B7&9)&4!'.!5*)%!Q)<OYO<"!

I6*+!OP'.&!<M!'14!Q)$OWU!I6*+!OP'.&!WM!%'3&!5&&1!.%*,1!)*!9*;7/&;&1)!Q)<OYO<#!'14!

Q)$OW#8!+&.7&9)-3&/0!I\*5&+)!&/!'/>8!"_U_j!\();'11!&)!'/>8!"_U"!'14!H'1-1*!&)!'/>8!"__bM>!

$%&.&!7/'1).!,-//!5&!(.&4!)*!6(+)%&+!-13&.)-:')&!-6!9/&'3':&!*6!Q)GQAU!$DS!50!OP'.&!<!-.!

6*//*,&4!50!OP'.&!W8!)*!:&1&+')&!'!1&,!1*1J9*4-1:!OPQ>!Q.!4*(5/&!`1*92!*()!*6!1(9/&'+!

OP'.&!<!*+!OP'.&!W!-1!84#0(9/-)()! -.!/&)%'/8!)%&.&!/-1&.!'+&!1&9&..'+0!-1!*+4&+!)*!4*,1!

+&:(/')&!'44-)-*1'//0!5*)%!Q)<OYO<"!'14!Q)$OWU8!6*+!&B';7/&!50!KL\S>!

!

g? F.7.:/-&.$%*/B*A.$.%(<*%//:)*%/*($7.)%(A#%.*%3.*B"$<%(/$*/B*8%H8=I*GR'*($*7(7/*

!L1!*+4&+!)*!6(+)%&+!-13&.)-:')&!)%&!5-*/*:-9'/!6(19)-*1.!*6!Q)GQAU8!,&!*+4&+&4!'14!3&+-6-&4!

'!$J=PQ!/-1&!6+*;!)%&!\'5-J`')!9*//&9)-*1>!$%&!/-1&!_#UHU_!,'.!'!3&+-6-&4!\'2-J`')!$J=PQ!

-1.&+)-*1!/-1&!'14!,&!9*16-+;&4!50!:&1*)07-1:!'14!O$J<HO!)%')!)%-.!/-1&!%'4!-14&&4!'!$J

=PQ!-1.&+)-*1!-1!Q)GQAU!IA-:(+&!T_M>!L)!-.!-1)&+&.)-1:!)*!1*)&!)%')!)%-.!/-1&!4*&.!1*)!%'3&!

'10!.7&9-6-9!;'9+*.9*7-9!7%&1*)07&>!A-1'//08!'6)&+!.&R(&19-1:!*6!)%&!+&:-*1!,%&+&!)%&!$J

=PQ!,'.!-1.&+)&4!,&!9*16-+;&4!)%&!$J=PQ!-1.&+)-*1!IA-:(+&!T_M>!$%&1!)*!-13&.)-:')&!-1!

;*+&!4&)'-/.!)%&!5-*/*:-9'/!6(19)-*1I.M!*6!Q)GQAU!&3*/()-*1'+0!9*1.&+3&4!$DS!,&!9+&')&4!

1&,!:&1&)-9'//0!;*4-6-&4!Q)GQAU!/-1&.>!A*+!)%-.8!'!"!257!!

!

! U"[!

!

!

!

F2*L0(.YCX!?R303",(02-3,2%$.%W.!,+3WT.G3/2I!<.C6T?TC.<A5@!.2$-(0,2%$.'2$(>!S9%&;')-9!-1.&+)-*1!*6!

)%&! $J=PQ! -1* 8%&#BI! :&1&! -.! .%*,1! I(77&+! 7'+)M>! $J=PQ! -1.&+)-*1! &B'9)! /*9'/-@')-*1! ,'.! 3&+-6-&4! 50!

.&R(&19-1:>!L1.&+)-*1!*6!)%&!$J=PQ!/-1&!,'.!3&+-6-&4!50!<HO>!\`!.)'14!6*+!\'5-!`')8!]$!6*+!]-/4!$07&!'14!

C! 6*+!5/'12>!=PQ!;*/&9(/'+!,&-:%)!;'+2&+.!'+&! -14-9')&4!50!1(;5&+.! IQM>!8%&#BI!`1*92!Y()!,'.!'/.*!

3&+-6-&4!50!O$J<HO>!Q6)&+!+&3&+.&!)+'1.9+-7)-*1!*1!)*)'/!OPQ8!Q)GQAU!H=S!,'.!';7/-6-&4!(.-1:!7+-;&+.!')!

5*)%!&B)+&;-)-&.>!GQAU!H=S!9*(/4!5&!';7/-6-&4!-1!)%&!]$!IH*/JYM!5()!1*)!-1!)%&!\'5-J`')!_#UHU_!/-1&>!L1!

the “–RT” lane, the reverse transcription mix + RNA but without the reverse transcriptase enzyme was used

as a template for PCR. In the blank lane “B”, only water was used!'.!<HO!)&;7/')&!ICM>!

!

! !

! U"g!

+&:-*1!(7.)+&';!*6!)%&!GQAU!:&1&!,'.!6-+.)!';7/-6-&4!I'.!)%&!&B'9)!7+*;*)&+!.&R(&19&!*6!

Q)GQAU!-.!(121*,1M!,-)%!)%&!6(//!/&1:)%!:&1*;-9!3&+.-*1!*6!)%&!:&1&!'14!-1)+*4(9&4!-1!

'1!&1)+0!3&9)*+!I%&+&!7=*1*+"_gM!(.-1:!)%&!\')&,'0!i!)&9%1*/*:0>!$%&1!;()':&1&.-.!

was performed to remove either the TLS (Δ TLS) or the entire intron containing the TLS

(Δ i6). A version with only AtMQAU!9*4-1:!.&R(&19&!IH=SM!,'.!'/.*!9+&')&4!(14&+!)%&!

9*1)+*/! *6! '! .)+*1:! 7+*;*)&+! I#^S!Q)GQAUM! )*! -13&.)-:')&! )%&! &66&9)! *6!Q)GQAU! *3&+J

&B7+&..-*1! IA-:(+&! TUM>! A-1'//08! )%&.&! )%+&&! 3&+.-*1.! I'14! '1! (1;*4-6-&4! 3&+.-*1! *6!

Q)GQAU!,-)%!"!257!*6!)%&!(7.)+&';!:&1*;-9!+&:-*1!I)%')!,-//!5&!(.&4!'.!'!7*.-)-3&!9*1)+*/!

)*! 9*;7/&;&1)! )%&! &#BI! `Y! /-1&.MM! ,&+&! 9/*1&4! -1! 4&.)-1')-*1! 3&9)*+.! '148! '6)&+!

)+'1.6*+;')-*1!-1!8A4/0#<%.4("&*%"&.B#<(.$)!I\K!#U_UM8!&#BI*`Y!7/'1).!I\'5-!_#UHU_M!

,&+&! )+'1.6*+;&4! (.-1:! )%&! 6/*+'/! 4-7! ;&)%*4>! </'1).! &66&9)-3&/0! )+'1.6*+;&4! ,&+&!

.&/&9)&4!(.-1:!)%&!C'.)'!%&+5-9-4&>!</'1).!9*1)'-1-1:!)%&!C'.)'!%&+5-9-4&!+&.-.)'19&!CQO!

:&1&! I'14! 9*1.&R(&1)/0! )+'1.6*+;&4M! ,&+&! +&.-.)'1)! )*! )%-.! )+&');&1)! '14! 6(+)%&+!

:&1*)07&!50!<HO!)*!;*1-)*+!)%&!-1.&+)-*1!*6!)%&!.&R(&19&!*6!-1)&+&.)!IA-:(+&!TUM>!$%&.&!

7/'1).!,-//!1&&4!)*!5&!/&4!)*!%*;*@0:*.-)0!)*!*5)'-1!.)'5/&!/-1&.>!

D-2&,-.&8!)%&!&#BI!\'5-`')!;()'1)!/-1&!4&.9+-5&4!'5*3&!,'.!'/.*!9+*..&4!,-)%!)%&!-4/4-^!

`Y!/-1&>!$%-.!-.!.)-//!*1:*-1:!,*+2!'.!7/'1)!%*;*@0:*(.!6*+!5*)%!)%&!GQAU!-1.&+)-*1!'14!

)%&!<OYO<#!-1.&+)-*1!.)-//.!1&&4.!)*!5&!-4&1)-6-&4!50!:&1*)07-1:!)%&!A"!:&1&+')-*1>!!

!

!

! U"f!

!

F2*L0(. YTN! -"R(+3,2". 0(P0(-($,3,2%$. %W. ,R(. )2WW(0($,. !,:!FT. 1(0-2%$-. "0(3,(). ,%."%+P'(+($,.1234.IJ.'2$(-!'14!-13&.)-:')&8!($*7(7/8!)%&!5-*/*:-9'/!6(19)-*1!*6!)%&!&3*/()-*1'+0!conserved TLS. In MAF1 Δ TLS, the TLS was removed. In MAF1 Δ I6 it is the intron containing the

$DS!)%')!,'.!+&;*3&4>!A-1'//08!'!3&+.-*1!9*1)'-1-1:!'!.)+*1:!7+*;*)&+!I#^SM!6(.&4!)*!Q)GQAU!H=S!,'.!:&1&+')&4!IGQAU!#^SM>!

!

!

F2*L0(.Y4N!G($%,DP2$*./D.;?8.%W.!,:!FT."%+P'(+($,3,2%$.'2$(-O!$*!&1.(+&!9*++&9)!-1.&+)-*1!*6!)%&!;*4-6-&4!3&+.-*1.!*6!Q)GQAU!-1!)%&!&#BI!2*!/-1&.8!<HO!,'.!7&+6*+;&4!*1!:&1*;-9!=PQ>!Q)GQAU!:&1&!9*(/4!1*)!5&!';7/-6-&4!-1!)%&!2*!/-1&.!I&#B!\`M8!,%&+&'.!-)!-.!';7/-6-&4!-1!)%&!2*!/-1&.!9*;7/&;&1)&4!,-)%!Q)!GQAU!,-)%*()!)%&!$DS!.&R(&19&!IJ$DSM8!,-)%*()!)%&!$DS!9*1)'-1-1:!-1)+*1!IJ-[M!*+!,-)%!)%&!6(//!/&1:)%!:&1&!.&R(&19&!'.!'!7*.-)-3&!9*1)+*/!IcADM>!]$!=PQ!IH*/JYM!-.!(.&4!'.!'!7*.-)-3&!9*1)+*/!'14!)%&!<HO!;-B!,-)%*()!)%&!=PQ!7*/0;&+'.&!ICM!'.!'!1&:')-3&!9*1)+*/>!

! U"b!

5? H8=I*)"0<.::":#4*:/<#:(L#%(/$*($*84#0(9/-)()*!A-1'//08!,&! -13&.)-:')&4!Q+'5-4*7.-.!GQAU!.(5J9&//(/'+! /*9'/-@')-*1!5&9'(.&!GQAU!%'.!

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.&R(&19-1:>!$%&!S'/2o_Uf^U"!$J=PQ!/-1&!9*1)'-1.!'!$J=PQ!-1.&+)-*1!-1!)%&!U_)%!-1)+*1!*6!

)%&!Q)$OGUQ!:&1&>!$%&!S'/2o"_[#fT!/-1&!9*1)'-1.!'!$J=PQ!-1.&+)-*1!-1!)%&!b)%!&B*1!*6!)%&!

Q)$OGUC! :&1&! '14! )%&!]-.9=.D*B"TT\_T! /-1&! 9*1)'-1.! '! $J=PQ! -1.&+)-*1! -1! )%&! b)%!

-1)+*1!*6!)%&!Q)$OGUC!:&1&>!

`1*92J*()!*6!)%&!4&.-+&4!:&1&!,'.!3&+-6-&4!50!O$J<HO!(.-1:!6*+,'+4!'14!+&3&+.&!7+-;&+.!

4&.-:1&4!)*!';7/-60!)%&!6(//!/&1:)%!9*4-1:!.&R(&19&!IH=SM>!O$J<HO!+&.(/).!.%*,!)%')!)%&.&!

/-1&.!9'1!&66&9)-3&/0!5&!9*1.-4&+&4!'.!8%%4&I#!'14!8%%4&I0!21*92*()!/-1&.!IA-:(+&!^^!'14!

^[M!>!

L)!-.!-1)&+&.)-1:!)*!1*)&!)%')*%4&I#!'14!%4&I0!21*92!*()!7/'1).!4-4!1*)!&B%-5-)!'10!*53-*(.!

7%&1*)07&>!

$%&! S'/2o_Uf^U"! /-1&! ,'.! 9+*..&4! ,-)%! )%&! ]-.9=.D*B"TT\_T! /-1&! '14! ,-)%! )%&!

S'/2o"_[#fT!)*!:&1&+')&!%4&I#!–!%4&I0!4*(5/&!;()'1).>!\&1*)07-1:!*6!)%&!A"!:&1&+')-*1!

-.! *1:*-1:! 6*+! )%&!]-.9=.D*B"TT\_T! B!S'/2o_Uf^U">! C()! 1*! 4*(5/&!;()'1).! 9*(/4! 5&!

-4&1)-6-&4!.*!6'+!I1a"^M>!H*19&+1-1:!)%&!9+*..!5&),&&1!)%&!S'/2o_Uf^U"!'14!S'/2o"_[#fT!

/-1&.8!)%&!A"!:&1&+')-*1!,-//!.**1!5&!:&1*)07&4>!

! U^T!

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F2*L0(.ZZX.8<A;?8.%$.!,<8:T!.*($(!using primers at the 5’ and 3’ end of the coding sequence (CDS). The

+&.(/)!9*16-+;.!)%')!$OGUQ!6(//J/&1:)%!)+'1.9+-7)!-.!1*)!&B7+&..&4!-1!)%&!S'/2o_Uf^"U"!/-1&>!

!

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F2*L0(.Z[X.8<A;?8.%$.!,<8:T=.*($(!using primers at the 5’ and 3’ end of the coding sequence (CDS). The

+&.(/)!9*16-+;.!)%')!$OGUC!6(//J/&1:)%!)+'1.9+-7)!-.!1*)!&B7+&..&4!-1!)%&!S'/2o"[#fT!'14!]-.94.D*B"TT\YT!

/-1&>!

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GQAU8!)%&!;'-1!1&:')-3&!+&:(/')*+!*6!OPQ!7*/0;&+'.&!LLL8!9*1)'-1.!-1!7/'1).8!'!)OPQ!/-2&!

S)+(9)(+&!I$DSM!-1!-).!:&1&)-9!.&R(&19&!9*1.&+3&4!-1!'//!)%&!'%4.-%/-3D%#*:&1*;&!'3'-/'5/&!

)*! 4')&>! S-19&! '%4.-%/-3D%#! '14! 13:/4/-3D%#! '+&! 9*1.-4&+&4! )*! %'3&! .7/-))&4! -1! ),*!

&3*/()-*1'+0! /-1&':&.! 6+*;! )%&!M(4(9(-:#$%#.! 5&),&&1!g"^!'14!U"__!;-//-*1!0&'+.!':*!

IC&92&+!'14!G'+-18!"__bM8!,&!%07*)%&.-@&4!)%')!)%-.!1*1!9*4-1:!OPQ!.&R(&19&!.%*(/4!

7/'0!'1!-;7*+)'1)!+*/&!-1!7/'1)!5-*/*:0!)%')!,*(/4!&B7/'-1!-).!&3*/()-*1'+0!9*1.&+3')-*1>!

C&9'(.&!OP'.&!<!-.!'1!&1@0;&!21*,1!)*!9/&'3&!5*)%!7+&9(+.*+!)OPQ.!'14!$DS!-1!3-3*!

I\*5&+)! &)! '/>8"_U_! '14! \();'11! &)! '/>! "_U"M! ,&! 4&9-4&4! )*! -13&.)-:')&! -6! GQAU!

&3*/()-*1'+0!9*1.&+3&4!$DS!9*(/4!5&8!-1!84#0(9/-)()8!'!1&,!.(5.)+')&!*6!)%&!1(9/&'+!OP'.&!

<!!&1@0;&.!<OYO<"d#>!

]&!7+*3&4!50!($*7(%4/!9/&'3':&!'..'0.!)%')!Q)<OYO<"!9'1!9/&'3&!Q)GQAU!-1)+*1-9!$DS8!')!

-).!&B7&9)&4!.)'+)!.-)&8!'.!&3-4&19&4!50!*(+!9O$J<HO!+&.(/).>!G*+&*3&+8!,&!-13&.)-:')&4!

)%&!&66&9)!*6!Q)<OYO<"d#!4*,1!+&:(/')-*1!*1!)%&!/&3&/!*6!Q)GQAU!')!)%&!)+'1.9+-7)*;-9!

/&3&/! '14! .%*,&4! )%')! 4&7/&)-*1! *6! Q)-4/4-Tf^* /&'4.! )*! '1! '99(;(/')-*1! *6! Q)GQAU!

)+'1.9+-7).>!Q)!/'.)8!,&!.%*,&4!)%')!($*7(7/8!Q)GQAU!)+'1.9+-7).!)&+;-1-!4*!'99(;(/')&!')!

)%&! &B'9)! .)'+)! .-)&! *6! )%&! $DS! '14! )%')! )%&.&! .7&9-6-9! )+'1.9+-7).! %'3&! '44-)-*1'/!

nucleotides added in 3’8!,%-9%!.(::&.).!)%')!)%&0!,-//!5&!4&:+'4&4>!!

!

;%--2/'(.3',(0$3,21(.WL$",2%$-.W%0.:!FT.<K9.!Y(+!;'-1!%07*)%&.-.!7+*7*.&.!)%')!)%&!9*1.&+3&4!GQAU!$DS!.&+3&.!'.!'!.-:1'/!)*!9/&'3&!

GQAU!;OPQ!'14!)%(.!)*!9*1)+*/!-).!/&3&/>!S)-//8!#0*($(%(/8!'/)&+1')-3&!%07*)%&.&.!9*(/4!5&!

&13-.':&4!)*!&B7/'-1!)%&!6(19)-*1!*6!GQAU!$DS>!L1!'!6-+.)!%07*)%&.-.8!OP'.&!<!9/&'3':&!*6!

GQAU!$DS!9*(/4!5&! 6*//*,&4!50!OP'.&!W!9/&'3':&!)*!:&1&+')&!'! 6(19)-*1'/!1*1J9*4-1:!

OPQ>! Q/)&+1')-3&/08! )%&! *5.&+3&4! 9/&'3':&! *6! GQAU! $DS! 9*(/4! %'3&! 5&&1! 4(&! )*!

)+'1.9+-7)-*1!&/*1:')-*1!6'9)*+.!'.!4&.9+-5&4!6*+!84#0(9/-)()!-1!Q1)*.@!&)!'/>!"_"_8!-6!OPQ!

7*/0;&+'.&!LL!%'4!%'4!'!)&14&190!)*!.)'//!')!)%&!.7&9-6-9!$DS!/*9(.>!A-1'//08!-)!9*(/4!'/.*!5&!

%07*)%&.-@&4! )%')8! .-19&!Q)GQAU!$DS! -.! /*9')&4! -1! '1! -1)+*18! )%-.! 9*1.&+3&4! .)+(9)(+&!

! U^[!

,*(/4! -1! 6'9)! 5&! +&/')&4! )*! .7/-9-1:! I*+! '/)&+1')-3&! .7/-9-1:M>! ?*,&3&+8! )%&! +&.(/).! *6!

-13&.)-:')-*1.! 7+&.&1)&4! -1! )%-.! )%&.-.! 4*! 1*)! 7+*3-4&! &3-4&19&.! )%')! ,*(/4! '//*,! )*!

.(77*+)!*1&!*6!)%&.&!'/)&+1')-3&!%07*)%&.&.>!

L14&&48!*(+!-13&.)-:')-*1!*1!7/'1)!21*92&4!*()!6*+!Q%&#BI!'14!9*;7/&;&1)&4!50!GQAU!

:&1&!,-)%*()! )%&!$DS!.&R(&19&!*+!,-)%*()! )%&! -1)+*1!9*1)'-1-1:!Q)GQAU!$DS8!.%*,&4!

)%')!Q)GQAU!;OPQ!-.!.7/-9&4!9*++&9)/0!5&9'(.&!*(+!O$J<HO!&B7&+-;&1)!7&+6*+;&4!*1!

OPQ!&B)+'9)&4!6+*;!)%&.&!)+'1.:&1-9!/-1&.!.%*,&4!)%')!)%&!O$J<HO!7+*4(9).!*5)'-1&4!%'4!

)%&!.';&!.-@&!'.!)%&!]$!IH*/JYM>!G*+&*3&+8!Q)GQAU!$DS!'77&'+.!)*!5&!)%&!*1/0!-1)+*1-9!

19OPQ! 9*1.&+3&4! ,-4&/0! -1! /'14! 7/'1).! IQ22(+')*3! &)! '/>8! "_UTM>! L6! )%&! -1)+*1-9! $DS!

6(19)-*1!%'4!5&&1!+&/')&4!)*! .7/-9-1:8! -)!,*(/4!%'3&!5&&1! /-2&/0!)%')! .-;-/'+!.)+(9)(+&.!

,*(/4!*99(+!-1!;*+&!)%'1!*1&!*6!)%&!"^!___!:&1&.!&19*4&4!-1!)%&!84#0(9/-)()!:&1*;&8!

;'10!*6!,%-9%!%'3-1:!;(/)-7/&!-1)+*1.>!

Q/.*8!)%&!'3'-/'5/&!H%-7JS&R!4')'!*5)'-1&4!,-)%!'1)-5*4-&.!':'-1.)!)%&!HJ)&+;-1'/!4*;'-1!

*6!Q+'5-4*7.-.!OPQ!7*/0;&+'.&! LL8! )%')! 9'1!5&! 9*1.-4&+&4!'.! +&7+&.&1)-1:!OPQ!<*/>! LL!

*99(7'190!($*7(7/8!4*!1*)!.%*,!'10!*53-*(.!.-)&!,%&+&!)%&!7*/0;&+'.&!,*(/4!5&!.)'//-1:!

*1!)%&!GQAU!:&1&>!L14&&48!)%&!4-.)+-5()-*1!*6!)%&!+&'4.!-1!)%&!4')'!6+*;!Q1)*.@!&)!'/>!"_"_!

-.! %*;*:&1&*(.! ')! )%&!Q)GQAU! /*9(.! IQ)^:U#"T_M8! -14-9')-1:! )%')! OPQ!7*/0;&+'.&! LL!

.)'//-1:! ')! )%&!$DS! .-)&! '.! '! +&:(/')-*1! *6!Q)GQAU! /&3&/! *6! &B7+&..-*1! -.! 3&+0! (1/-2&/0>!

H*1.&R(&1)/08! -)! is thus unlikely that the cleaved transcripts detected in our 3’RACE

'..'0.!I*+! -1!)%&!7(5/-9/0!'3'-/'5/&!Q+'5-4*7.-.!<QOZJ.&R!=')'M!'+&!:&1&+')&4!50!OPQ!

7*/0;&+'.&!LL!+')%&+!)%'1!50!9/&'3':&!50!Q)<OYO<"d#>!

!

?%L').8@3-(.Q./(.2$1%'1().2$.,R(.P0%)L",2%$.%W.3.$%1('.$%$."%)2$*.8@!_.!Q.!;&1)-*1&4!'5*3&8!'1!'/)&+1')-3&!%07*)%&.-.!9*(/4!5&!)%')!'6)&+!9/&'3':&!50!OP'.&!<8!

AtMAF1 TLS is also cleaved by RNase Z (the enzyme catalysing the removal of the 3’ trailer

.&R(&19&!6+*;!)OPQM!)*!:&1&+')&!'!6(19)-*1'/!.;'//!OPQ>!$%-.!%07*)%&.-.!-.!9&+)'-1/0!)%&!

;*.)!-1)&+&.)-1:!'/)&+1')-3&!%07*)%&.-.!)*!)%&!*1&!,%&+&!9/&'3':&!50!OP'.&!<!*1/0!/&'4.!

)*!Q)GQAU!7+&J;OPQ!4&:+'4')-*1!'14!'.! .(9%!,*(/4!5&!'!7*.)J)+'1.9+-7)-*1'/!4*,1J

+&:(/')-*1!;&9%'1-.;!)*!6-1&!)(1&!GQAU!&B7+&..-*1!-1!/'14!7/'1).>!!

It must be stated first that the occurrence of a cleavage in 3’ of AtMAF1 TLS by RNase Z is

1*)! 1&9&..'+-/0! 9*1)+'4-9)*+0!,-)%!*(+!;'-1! %07*)%&.-.! 5&9'(.&! )%&! 9/&'3':&! 50! 5*)%!

! U^g!

OP'.&!<!'14!OP'.&!W!)*!:&1&+')&!'!1&,!6(19)-*1'/!19OPQ!9*(/4!')!)%&!.';&!)-;&!'/.*!/&'4!

)*!-;7'-+&4!.7/-9-1:!'14!)%&!4&:+'4')-*1!*6!Q)GQAU!7+&J;OPQ>!S-;-/'+!9'.&.!*6!9/&'3':&!

*6!'!$DS!)*!:&1&+')&!'!1*1J9*4-1:!OPQ!%'3&!'/+&'40!5&&1!4&.9+-5&4!-1!)%&!/-)&+')(+&>!$%&!

;*.)!6';*(.!&B';7/&.!-1!Z(2'+0*)&.!'+&!)%*.&!*6!)%&!)OPQ! /-2&!19OPQ.!;'.9OPQ!'14!

Men β. These two ncRNA are processed from, respectively the lncRNA MALAT1 and MEN

β. According to the published articles, MEN β tRNA like small RNA has a CCACCA motif

added at its 3’ end that will serve as a signal for its rapid deg+'4')-*1>!$%-.!,*(/4!&B7/'-1!

,%0!)%-.!19OPQ!-.!(14&)&9)'5/&!50!'!P*+)%&+1!5/*)!'77+*'9%!IS(1,**!&)!'/8!"__bj!]-/(.@!

&)! '/8! "_UUj! `(%1! &)! '/8"_U^M>! L1! 9*1)+'.)8! )%&!;'.9OPQ! :&1&+')&4! 6+*;! )%&!GQDQ$U!

/19OPQ! -.! '1! -1)&+&.)-1:! 9'.&!*6!'! 6(19)-*1'/! )OPQJ/-2&!1*1!9*4-1:!OPQ!'.! -)!%'.!5&&1!

.%*,1!)*!(14&+:*!HHQ!'44-)-*1!I]-/(.@!&)!'>8!"__fM!'14!3&+0!+&9&1)/08!)*!7+*;*)&!:/*5'/!

7+*)&-1! )+'1./')-*1! '14! 9&//! 7+*/-6&+')-*1! 50! 7*.-)-3&/0! +&:(/')-1:! )%&! 7+*)&-1! /&3&/! *6!

:/()';-10/J)OPQ!.01)%&)'.&!!IlQOSM!ID(!&)!'/>8"_"_M>!

?*,&3&+8!&3&1!-6!)%&!%07*)%&.-.!*6!'!9/&'3':&!50!OP'.&!W!6*//*,-1:!OP'.&!<!9/&'3':&!I'.!

*(+!($*7(%4/!9/&'3':&!'..'0.!'14!)%&!9(++&1)!.)')&!*6!21*,/&4:&!-14-9')&!)%')!)%-.!*+4&+!-.!

)%&! ;*.)! /-2&/0M! -.! -1)&+&.)-1:! '14! 9'11*)! 0&)! 5&! +(/&4! *()8! ,&! 4-4! 1*)! 6-14! '10!

&B7&+-;&1)'/! &3-4&19&! )*! .(77*+)! -)>! L14&&48! )%&+&! -.! 6*+! )%&! ;*;&1)8! 1*! 7+**6! )%')!

Q)GQAU! 9/&3':&! 50! OP'.&! <! /&'4.! )*! )%&! 9+&')-*1! *6! '! 1&,! .;'//! 1*1J9*4-1:! OPQ!

I.19OPQM!-1!Q+'5-4*7.-.!($*7(7/>!L1!7'+)-9(/'+8!*(+!($*7(%4/!'..'0.!.%*,.!)%')!Q)OP'.&!W#!

I$OW#M! /-2&/0! 9/&'3&.! *1/0! '6)&+! OP'.&! <! 9/&'3':&8! 5()! ,&! 9*(/4! 1*)! -4&1)-60! .7&9-6-9!

AtRNaseZ3 cleavages in our 3’Race assays nor in the publicly available PAREJ.&R!4')'>!

A(+)%&+;*+&8!1*! .;'//!OPQ!9*++&.7*14-1:! )*!)%&!$DS! .&R(&19&!9*(/4!5&! -4&1)-6-&4!50!

P*+)%&+1J5/*)!50!Q22(+')*3!&)!'/>!*+!-1!)%&!4-66&+&1)!7(5/-9!4')'5'.&.!*6!.;'//!OPQ!)%')!

,&! '1'/0.&4>! P&3&+)%&/&..8! '5.&19&! *6! 7+**6! -.! 4&6-1-)-3&/0! 1*)! '! 7+**6! *6! '5.&19&>!

H*1.&R(&1)/08!,&!,-//!1&&4!)*!6(+)%&+!-13&.)-:')&!)%-.!7*..-5-/-)0!*6!'1!OP'.&!W!9/&'3':&!

($* 7(7/. One way to investigate this question is to perform comprehensive 5’RACE

analyses. The 5’RACE that I performed at the beginning of my PhD identified a few

.&R(&19&.! )%')! 9*(/4! 9*++&.7*14! )*! '! 9/&'3':&! *6! Q)GQAU! $DS! 50! OP'.&! W8! 5()! '.!

&B7/'ined above in the Results section, this 5’RACE assay was performed with an RNA

'4'7)&+!)%')!4-4!1*)!'//*,!)*!4-66&+&1)-')&!5&),&&1!<HO!4(7/-9')&.!'14!(1-R(&!OPQ!+&'4.>!

Consequently, performing 5’RACE with an RNA adapter containing a random region to

-4&1)-60! (1-R(&! +&'4.! 6+*;!<HO!4(7/-9')&.! .&&;.! )*! 5&! )%&!;*.)! '4&R(')&!;&)%*4! )*!

-13&.)-:')&!)%*+*(:%/0!-6!'1!OP'.&!W!9/&'3':&!*6!Q)GQAU!$DS!4*&.!!*99(+!($*7(7/!)*:&)%&+!

! U^f!

,-)%!)%&!9/&'3':&!50!OP'.&!<>!Q)!)%-.!.)':&!,&!)%(.!9'11*)!+(/&!*()!)%')!GQAU!$DS!;-:)%!

5&! +&/&'.&4! 6+*;! -).! -1)+*1! '14! )%')! -)! 9*(/4! '99(;(/')&! &3&1! ')! /*,! /&3&/.! 6*+! '! 0&)!

(1-4&1)-6-&4!6(19)-*1>!

!

B$)20(",.(WW(",.%W.:!FT."'(313*(.%$.8@!.P%'.BBB.3",212,D.!L1! *(+! ;*4&/8! ,%&+&! <OYO<! 4*,1J+&:(/')-*1! +&.(/).! -1! GQAU! -19+&'.&4! /&3&/8! ,&!

7+&4-9)&4!)%')!)%&!)+'1.9+-7)-*1!*6!'//!OPQ.!)+'1.9+-5&4!50!OPQ!7*/>!LLL!9*(/4!5&!-;7'-+&4>!

?*,&3&+8!6*+!1*,!*18!,&!9*(/4!1*)!4&)&9)!'!.-:1-6-9'1)!4&9+&'.&!*6!)%&!/&3&/.!*6!OPQ!7*/>!

LLL!)+'1.9+-7).!.(9%!'.!E[!.1OPQ!*+!gSD!OPQ.!,%-/&!7+&3-*(.!,*+2!%'4!.%*,1!)%')!)%&!

/&3&/! *6! ;')(+&! 1(9/&'+! )OPQ! -.! 4&9+&'.&4! -1! -4/4-^! `Y! ;()'1).! ,%&+&! <OYO<"! -.!

4*,1+&:(/')&4!I\();'11!&)!'/>!"_U"M>!$%-.!&66&9)!*1!)OPQ!/&3&/.!9*(/4!5&!4-+&9)/0!4(&!)*!

'!4&9+&'.&!-1!7+&9(+.*+J)OPQ!;')(+')-*1!I4(&!)*!)%&!/*,&+!';*(1)!*6!OP'.&!<!&1@0;&M!

'14!d!*+!)*!*(+!7+*7*.&4!+&:(/')-*1!/**7>!L1!'//!9'.&.8!OPQ!7*/>!LLL!)+'1.9+-7).!/&3&/.!'+&!

1*)!'//!'66&9)&4!50!<OYO<!4*,1J+&:(/')-*1!.-19&!^S!+OPQ!/&3&/.!3'+-')-*1.!,&+&!1&3&+!

*5.&+3&48!1&-)%&+!!-1!)%&!7+&.&1)!.)(408!1*+!-1!7+&3-*(./0!7(5/-.%&4!,*+2!I\();'11!&)!'/>!

"_U"M>!P*1&)%&/&..8!)%&!'5.&19&!*6!OPQ!.)&'40!.)')&!/&3&/!3'+-')-*1.8!'.!*5.&+3&4!%&+&!6*+!

E[!.1OPQ!!*+!gSD!OPQ.!4*&.!1*)!7+*3&!)%')!OPQ!7*/>!LLL!)+'1.9+-7)-*1!,'.!1*)!+&:(/')&4>!

L)! -.! -;':-1'5/&! )%')! )%&! )(+1*3&+!*+! 4&:+'4')-*1! +')&! *6! .7&9-6-9! )+'1.9+-7).!;-:%)! 5&!

+&:(/')&4!'.!,&//!-6!OPQ!7*/>!LLL!)+'1.9+-7)-*1!-.!4&9+&'.&48!->&>!)%')!)+'1.9+-7).!.(9%!'.!^S!

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indiquent que l’ordre le!#%6!0#!105%!1$'A*A0#!est celui d’un clivage par la RNase

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Z3 (TRZ3) ne clive probablement qu’après le clivage de la RNase P, mais nous

n’avons pas pu identifier de clivages spécifiques d’AtRNaseZ`! 8*(%! ('%!

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:;*%#!c! ,-./,/&. L’une des façons d’étudier cette question consiste à effectu#$!

des analyses de 5’RACEL! I*! U9:-MQ! O5#! P9*&! $3*0&%3#! *5! 83A56! 8#!=*! 6?Y%#! *!

&8#(6&7&3!O5#0O5#%!%3O5#(2#%!O5&!1'5$$*&#(6!2'$$#%1'(8$#!B!5(!20&N*4#!85!HIJ!

d’AtMAF1! 1*$! 0*! :;*%#! c/! =*&%! 2'==#! #R10&O53! 2&D8#%%5%! 8*(%! 0*! %#26&'(!

:3%506*6%/! 2#! 6#%6! U9:-MQ! *! 363! $3*0&%3! *N#2! 5(! *8*16*6#5$! -:;! O5&! (#!

1#$=#66*&6! 1*%! 8#! 8&773$#(2&#$! 0#%! 8'5A0'(%! 8#! \M:! 8#%! %3O5#(2#%! 89-:;!

5(&O5#%L! \ar conséquent, la réalisation d’expérience de! U9:-MQ! *N#2! 5(!

*8*16*6#5$!89-:;!2'(6#(*(6!5(#!$34&'(!*03*6'&$#!1'5$!&8#(6&7&#$!0#%!%3O5#(2#%!

5(&O5#%!#6!0#%!8&773$#(2&#$!8#!8'5A0'(%!8#!\M:!%#=A0#!^6$#!0*!=36?'8#!0*!105%!

*83O5*6#!1'5$!3658&#$!8#!=*(&Y$#!*11$'7'(8&#!%&!5(!20&N*4#!1*$!0*!:;*%#!c!8#!

-6,-).!HIJ!%#!1$'85&6!,-./,/&!*1$3%!0#!20&N*4#!1*$!0*!:;*%#!\L!-&(%&/!B!2#!%6*8#/!

('5%! (#! 1'5N'(%! 8'(2! 1*%! #R205$#! O5#! 0#! HIJ! 8#! -6,-).! %'&6! 0&A3$3! 8#! %'(!

&(6$'(! #6! O59&0! 1'5$$*&6! %9*225=50#$! =^=#! B! 8#! 7*&A0#%! (&N#*5R! 1'5$! 5(#!

7'(26&'(!#(2'$#!('(!&8#(6&7&3#L!

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Discussion relative à l’effet éventuel sur l’activité de la Polymérase

1!;45!???@!7&!%+#0-2/!7&!89:!7/!;,<;=>!3-)!;,A4B4AC!/,!D!

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a*(%!('6$#!='8Y0#/!'f! 0*!$3450*6&'(!B! 0*!A*&%%#!8#!\:_:\T!#6!`!#(6$*o(#!5(#!

*54=#(6*6&'(!85!(&N#*5!8#! l’expression de!-6,-)./!('5%!*N'(%!1$38&6!O5#!0*!

6$*(%2$&16&'(!8#! 6'5%! 0#%!-:;! 6$*(%2$&6%! 1*$! 09-:;!1'0L! >>>! 1'5$$*&6! ^6$#!*063$3L!

M#1#(8*(6/! 1'5$! 09&(%6*(6/! ('5%! (9*N'(%! 1*%! 15! 836#26#$! 5(#! 8&=&(56&'(!

%&4nificative des niveaux d’expression d#%!6$*(%2$&6%!de l’ARN polymérase >>>!6#0%!

O5#!C!dp! %(:;-!F!'5!#(2'$#! 0#!C!SJI!-:;!F!*0'$%!O5#!8#%! 6$*N*5R!*(63$&#5$%!

*N*&#(6!='(6$3! O5#! 0#! (&N#*5! 89-:;6! (5203*&$#!=*65$#! #%6! 8&=&(53! 2?#@! 0#%!

=56*(6%!n’exprimant plus AtPRORP3 («!%#&#%7.89!FK!#6!'f!-6\:_:\T!#%6!$34503!

B!0*!A*&%%#!G]56=*((!#6!*0LTV.TKL!M#6!#77#6!%5$!0#%!(&N#*5R!89-:;6!1'5$$*&6!^6$#!

8&$#26#=#(6!8e!B!5(#!8&=&(56&'(!8#!0*!=*65$*6&'(!85!1$325$%#5$!8#%!-:;6!G#(!

$*&%'(!8#! 0*!O5*(6&63! &(73$&#5$#!89#(@<=#!:;*%#!\K!#6! b!'5!B!('6$#!A'520#!8#!

$3450*6&'(!1$'1'%3#!8*(%!0*!1*$6&#!$3%506*6!8#!2#66#!6?3%#L!a*(%!6'5%!0#%!2*%/!0#%!

(&N#*5R!8#!6$*(%2$&16&'(!8#%!1$&(2&1*5R!-:;!6$*(%2$&6%!1*$!09-:;!1'0L!>>>!(#!%'(6!

1*%! 6'5%! *77#263%! 1*$! 0*! $3450*6&'(! B! 0*! A*&%%#! 8#%! \:_:\! (5203*&$#%!

d’0#)1,2&%3,3! 15&%O5#! 0#%! N*$&*6&'(%! 8#%! (&N#*5R! 89-:;$! UJ! (9'(6! P*=*&%! 363!

'A%#$N3#%/! (&! 8*(%! 0*! 1$3%#(6#! 3658#/! (&! 8*(%! 8#%! 6$*N*5R! 15A0&3%!

*(63$&#5$#=#(6!G]56=*((!#6!*0L!TV.TKL!;3*(='&(%/!09*A%#(2#!8#!N*$&*6&'(%!8#%!

(&N#*5!d’expression de ces -:;!B!0936*6!893O5&0&A$#/!2'==#!'A%#$N3!&2&!1'5$!dp!

%(:;-! '5! 1'5$! 0#! SJI! :;-! (#! 1$'5N#! 1*%! O5#! l’activité de transcription de

09-:;! 1'0L! >>>! (9#%6! 1*%! 83$34503#L! >0! #%6! #(! #77#6! &=*4&(*A0#! O5#! 0#! 6*5R! 8#!

$#('5N#00#=#(6!'5!8#!834$*8*6&'(!8#!6$*(%2$&6%!%132&7&O5#%!15&%%#!34*0#=#(6!

être régulé quand l’activité de! 09-:;!1'0L! >>>!#%6!8&=&(53#/! 29#%6DBD8&$#!O5#! 0#%!

6$*(%2$&6%!6#0%!O5#!0#%!-:;$!UJ!*5$*&#(6!*&(%&!5(#!8#=&DN&#!105%!0'(45#!0'$%O5#!

0#5$!6$*(%2$&16&'(!#%6!$385&6#L!

;3*(='&(%/!B!2#!%6*8#/!0#%!#77#6%!8&$#26%!'5!&(8&$#26%!8#!0*!$3450*6&'(!(34*6&N#!

8#!\:_:\!%5$! 09*26&N&63!8#! 09-:;!1'0<=3$*%#!>>>!$#%6#(6!B!2'=1$#(8$#L!q5%O59B!

1$3%#(6/!nous avons utilisé la méthode de la réduction de l’expression!43(&O5#!

&(85&6e par l’utilisation d’un! N&$5%! G">]JK!1'5$! $3450#$!B! 0*!A*&%%#! 0#%!#(@<=#%!

(5203*&$#%!8#!\:_:\!2?#@!0#)1,2&%3,3L!I#%!#77#6%!8#!2#66#!$3450*6&'(!B!0*!A*&%%#!

'(6!363!%5$N#&003%!1*$!:HDO\M:L!;'5%!*N'(%!34*0#=#(6!0*(23!5(#!*(*0<%#!:;-D

J#O!B!0932?#00#!85!6$*(%2$&16'=#!8#%!=56*(6%!%#&#%7.89!'f!\:_:\T!#%6!$34503!B!

0*! A*&%%#L! I#%! $3%506*6%! 1$30&=&(*&$#%! G('(! 1$3%#(63%! &2&K! 'A6#(5%! *N#2! 6$'&%!

$310&2*6%! A&'0'4&O5#%! (#! 6$'5N#(6! 1*%! 8#! 8&773$#(2#%! %6*6&%6&O5#=#(6!

%&4(&7&2*6&N#%! #(6$#! 0#%! =56*(6%! B! $3450*6&'(! (34*6&N#! #6! 0#%! 63='&(%L! M#0*!

%#=A0#! ^6$#! 8e! B! 0*! N*$&*A&0&63! #(6$#! 0#%! #R13$&#(2#%! ">]J! &(8&N&85#00#%L! a#!

=^=#/!8#%!8&773$#(2#%!7*&A0#%!85!(&N#*5!8#!6$*(%2$&16&'(!8#!2#$6*&(%!6$*(%2$&6%!

1'5$$*&#(6!^6$#!=*(O53#%!#(!$*&%'(!8#!0*!N*$&*A&0&63!&(?3$#(6#!*5R!#R13$&#(2#%!

8#!">]JL!a*(%!2#66#!'16&O5#/!*7&(!893658&#$!0*!O5#%6&'(!&=1'$6*(6#!8#!09*26&N&63!

8#! 09-:;! 1'0<=3$*%#! >>>! *5! 2'5$%! 8#! 0*! $3450*6&'(! (34*6&N#! 8#%! \:_:\!

nucléaires d’0#)1,2&%3,3/!#6!1*$!2'(%3O5#(6/! 09#77#6!8#! 0*!A'520#!8#!$3450*6&'(!

1$'1'%3#!8#! 09*26&N&63!8#! 09-:;!1'0<=3$*%#! >>>!N&*! 0#!20&N*4#!1*$! 0*!:;*%#!\!85!

HIJ! 2'(%#$N3! 89-6,-).!j! 8&773$#(6#%! *06#$(*6&N#%! #R13$&=#(6*0#%! 8'&N#(6! ^6$#!

#(N&%*43#%L! \*$! #R#=10#/! ('5%! 1'5$$&'(%! =#66$#! #(! 10*2#! 5(#! =36?'8#!

8&773$#(6#! 1'5$! 3658&#$! 09#77#6! 8#! 09*11*5N$&%%#=#(6! 8#! 0*! :;*%#! \! (5203*&$#!

2?#@! 0#)1,2&%3,3! O5&! %#$*&6! 105%! ?'='4Y(#/! 1*$! #R#=10#! #(! 56&0&%*(6!

09#R1$#%%&'(!8#!=&-:;!*$6&7&2&#0!'5!A*%3#!%5$!8#%!%<%6Y=#%!M:>J\:DM*%/!1*$!#RL!

#(! 56&0&%*(6! M*%.`! O5&! 2&A0#! 09-:;! GH#$(%! #6! *0L/! TV.mKL! d(#! *11$'2?#!

2'=103=#(6*&$#!1'5$$*&6!^6$#!8#!2$3#$!8#%!0&4(3#%!$*11'$6#5%#%!1'5$!09*26&N&63!

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`K!(5203*&$#L!M#1#(8*(6/!2#%!'56&0%!(#!%'(6!1*%!#(2'$#!8&%1'(&A0#%!1'5$!$3*0&%#$!

8#%! #R13$&#(2#%! ,-. /,/&L! \*$! #R#=10#/! 5(! 2'0'$*(6! 705'$'4Y(#! 1'5$! N&%5*0&%#$!

8#%!-:;!*!363!83N#0'113/!2'==#!832$&6!8*(%!5(!*$6&20#! $32#(6! GW'5?#88*!#6!

*0L/! TV.XKL! ,*&%! 2#66#! 6#2?(&O5#! (9#%6! 1*%! *8*163#! *5R! 8'%*4#%! ,-. /,/&L! I#!

83N#0'11#=#(6! 89'56&0%! %132&7&O5#%! %#$*! 8'(2! (32#%%*&$#! 1'5$! %5&N$#!

8&$#26#=#(6!0’activité de transcription de l’ARN pol. III et l’effet éventuel de la

dérégulation de l’expression de AtPROP2/3 sur cette 8#$(&Y$#L!

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\'5$! 2'(205$#! %5$! 2#66#! 1*$6&#/! &0! 2'(N&#(6! 8#! ('6#$! O5#! ,-).! #%6! 5(! 4Y(#!

&=1'$6*(6/!$3450*(6!09*26&N&63!8#!09-:;!1'0<=3$*%#!>>>/!#6!2'(%#$N3!2?#@!6'5%!0#%!

Eucaryotes. L’absence d’expression de MAF1! 8*(%! 0*! 0#N5$#! 2'(85&6! B! 5(#!

*225=50*6&'(!89-:;!8#!6$*(%7#$6!8*(%!0#%!2#0050#%!#6!B!5(#!2$'&%%*(2#!105%!0#(6#!

G\056*! #6! *0L/! T001). Cependant, l'absence d’expression de! ,-).! 8*(%! 0#%!

2#0050#%! *(&=*0#%! *77#26#! 0*! ='$1?'0'4&#! 2#0050*&$#! #6! 0*! 6*&00#! 2'$1'$#00#!

Gq'?(%'(! #6! *0L/! TVVSj! J?'$! #6! *0L/! TV.Vj! :&8#'56! #6! *0L/! TV.TK! 8#! =*(&Y$#!

2'(6$*%63#L!!

a#!105%/!36*(6!8'((3!O5#!09-:;!1'0L!>>>!%<(6?36&%#!8#%!-:;!('(!2'8*(6%!2$52&*5R!

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M#66#!$3450*6&'(!#%6!$3*0&%3#!1*$!,-).!#(!2*%!8#!%6$#%%!'5!8#! 0&=&6*6&'(!8#! 0*!

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>>>/! #=1^2?*(6! 0*! 7'$=*6&'(! 85! 2'=10#R#! 8#! 1$3D&(&6&*6&'(! G"t$0*(8#$! #6! *0L/!

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(5203*&$#! 89-$*A&8'1%&%! G-6\:_:\T! b! `K! 1#56! 20&N#$! 09-:;=! 1$325$%#5$!

d'AtMAF1 à un site spécifique situé sur l’ARN et conservé évolutivement/! O5&!

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RNAP III désigne l’ARN polymerase III. !

6a;-!0#%!4Y(#%!2'8*(6!1'5$!0#%!-:;!8#!6$*(%7#$6L!!

-6\T! *(8! -6H:c`! %'(6/! $#%1#26&N#=#(6/! 0#%! 4Y(#%! 8#! 0*! :;*%#! \! #6! 8#! 0*! :;*%#! c! 2?#@!

0#)1,2&%3,3L!!

Les points d’interrogation indiquent au lecteur les sujets sur lesquels des investigations

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H'56#%!0#%!36*1#%!&(8&O53#%!8*(%!2#!%2?3=*!%#!83$'50#(6!8*(%!0#!('<*5L!

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Afin d’étudier cette question/! ('5%! *N'(%! 15! %5&N$#! 0#%! #77#6%! 895(#! %3$&#! 8#!

%6$#%%! %5$! 8#%! 0&4(3#%! 8#! 10*(6#%!n’exprimant pas AtMAF1. Au cours de mon

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1. Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, et al. Rapid tRNA Decay Can Result from Lack of Nonessential Modifications. Molecular Cell. janv 2006;21(1):87- 96.

2. Anderson J, Phan L, Cuesta R, Carlson BA, Pak M, Asano K, et al. The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA. Genes & Development. 1 déc 1998;12(23):3650- 62.

3. Antosz W, Deforges J, Begcy K, Bruckmann A, Poirier Y, Dresselhaus T, et al. Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation. Plant Cell. mai 2020;32(5):1449- 63.

4. Antosz W, Deforges J, Begcy K, Bruckmann A, Poirier Y, Dresselhaus T, et al. Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation. Plant Cell. mai 2020;32(5):1449- 63.

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8. Awai T, Ochi A, Ihsanawati, Sengoku T, Hirata A, Bessho Y, et al. Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure. J Biol Chem. 7 oct 2011;286(40):35236- 46.

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12. Becker B, Marin B. Streptophyte algae and the origin of embryophytes. Annals of Botany. mai 2009;103(7):999- 1004.

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13. Bezanilla M, Perroud P - F., Pan A, Klueh P, Quatrano RS. An RNAi System in Physcomitrella patens with an Internal Marker for Silencing Allows for Rapid Identification of Loss of Function Phenotypes. Plant Biology. mai 2005;7(3):251- 7.

14. Blewett NH, Maraia RJ. La involvement in tRNA and other RNA processing events including differences among yeast and other eukaryotes. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. avr 2018;1861(4):361- 72.

15. Blewett NH, Maraia RJ. La involvement in tRNA and other RNA processing events including differences among yeast and other eukaryotes. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. avr 2018;1861(4):361- 72.

16. Boguta M, Czerska K, Żołądek T. Mutation in a new gene MAF1 affects tRNA suppressor efficiency in Saccharomyces cerevisiae. Gene. févr 1997;185(2):291- 6.

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l’extrémité 5’ des précurseurs d’ARN de transfert (ARNt). Mon travail de thèse a

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que d’identifier de nouveaux substrats ARN (autres que les ARNt) de PRORP2

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gènes. Enfin, le résultat principal de ma thèse est la mise en évidence d’un

nouveau substrat ARN de PRORP2. En effet, le transcrit d’un gène appelé MAF1

H1+'(!-)!@+'3(ion est de réguler l’activité de l’ARN polymerase III qui transcrit les

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thèse a permis de montrer que cette structure est conservée dans l’évolution

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une approche par 3’RACE a permis de montrer que des transcrits de MAF1

finissant juste avant cette structure s’accumulent %.* /%/'N! 8&! #$%,-()(! "&#A&(!

d’appuyer l’hypothèse que PRORP2 clive l’ARNm d&!76TR!%+/(!"+,#!0$'$#&#!,'!

"&(/(!6<;!'+'=codant, soit pour réguler le niveau d’expression de MAF1 et par

extension l’activité de l’ARN polymerase III. Globalement les résultats obtenus

au cours de cette thèse de doctorat ont permis d’améliorer notre conna/%%)'3&!

1&%!A+1)-/($%!1&!./+0$'M%&!1&%!6<;!1&!(#)'%@&#(!3E(+%+-/F,&%!3L&D!-&%!"-)'(&%!

)/'%/!F,&!1&!1$3+,5#/#!,'!'+,5&),!%,.%(#)(!6<;!1&!?<I<?JN!8&!1&#'/&#!#$%,-()(!

permet de proposer que PRORP2 pourrait réguler indirectement l’activité de la

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