Abstract Post-transcriptional processing of primary

transcripts can significantly affect both the quantity

and the structure of mature mRNAs and the corre-

sponding protein products. It is an important mecha-

nism of gene regulation in animals, yeast and plants.

Here we have investigated the interactive networks of

pre-mRNA processing factors in the developing grain

of wheat (Triticum aestivum), one of the world’s major

food staples. As a first step we isolated a homologue of

the plant specific AtRSZ33 splicing factor, which has

been shown to be involved in the early stages of em-

bryo development in Arabidopsis. Real-time PCR

showed that the wheat gene, designated TaRSZ38, is

expressed mainly in young, developing organs (flowers,

root, stem), and expression peaks in immature grain. In

situ hybridization and immunodetection revealed

preferential abundance of TaRSZ38 in mitotically ac-

tive tissues of the major storage organ of the grain, the

endosperm. The protein encoded by TaRSZ38 was

subsequently used as a starting bait in a two-hybrid

screen to identify additional factors in grain that are

involved in pre-mRNA processing. Most of the iden-

tified proteins showed high homology to known splic-

ing factors and splicing related proteins, supporting a

role for TaRSZ38 in spliceosome formation and 5¢ site

selection. Several clones were selected as baits in fur-

ther yeast two-hybrid screens. In total, cDNAs for 16

proteins were isolated. Among these proteins,

TaRSZ22, TaSRp30, TaU1-70K, and the large and

small subunits of TaU2AF, are wheat homologues of

known plant splicing factors. Several, additional pro-

teins are novel for plants and show homology to known

pre-mRNA splicing, splicing related and mRNA

export factors from yeast and mammals.

Keywords Grain development Æ Pre-mRNA

processing factors Æ Protein–protein interaction ÆSplicing Æ Yeast two-hybrid system

Abbreviations

AD Activation domain

BD Binding domain

DAP Days after pollination

dbEST GenBank expressed sequence tag

database

GUS b-Glucuronidase

NEB Nuclei extraction buffer

NLS Nuclear localization signal

NMD Nonsense mediated decay

RRM RNA recognition motif

RSZproteins Arginine/serine proteins with one or

two zinc fingers

SCL proteins SC35-like proteins

SD domains Serine/aspartic acid rich domains

snRNPs Small nuclear ribonucleoprotein

particles

Electronic Supplementary Material Supplementary materialis available to authorised users in the online version of thisarticle at http://dx.doi.org/10.1007/s11103-006-9046-6.

S. Lopato (&) Æ A. S. Milligan Æ N. Shirley ÆN. Bazanova Æ P. LangridgeAustralian Centre for Plant Functional Genomics,The University of Adelaide, PMB1, Glen Osmond,SA 5064, Australiae-mail: sergiy.lopato@acpfg.com.au

L. BorisjukInstitut fur Pflanzengenetik und Kulturpflanzenforschung,D-06466 Gatersleben, Germany

Plant Mol Biol (2006) 62:637–653

DOI 10.1007/s11103-006-9046-6

123

Systematic identification of factors involved in post-transcriptional processes in wheat grain

Sergiy Lopato Æ Ljudmilla Borisjuk ÆAndrew S. Milligan Æ Neil Shirley ÆNatalia Bazanova Æ Peter Langridge

Received: 8 March 2006 / Accepted: 6 July 2006 / Published online: 29 August 2006� Springer Science+Business Media B.V. 2006

SR domains Serine/arginine rich domains

TRN-SR Transportin-SR

Introduction

Comparative genome analyses indicate that increases

in gene number do not necessarily correlate with in-

creases in morphological and behavioral complexity

(Ewing and Green 2000). Pre-mRNA processing

(Reddy 2001; Quesada et al. 2003), transport (Lei and

Silver 2002), turnover (Abler and Green 1996) and

translational initiation (Kawaguchi and Bailey-Serres

2002) contribute additional sources of complexity.

Over the last two decades, numerous studies have

shown the importance in many organisms of these post-

transcriptional mechanisms of regulation, particularly

during embryogenesis (Sun et al. 1997; Lopez 1998;

Groth and Lardelli 2002; Kalyna et al. 2003; Lalli et al.

2003). Both the quantity and the structure of mature

mRNAs, and the corresponding protein products, can

be significantly affected by the post-transcriptional

processing of primary transcripts (Barrass and Beggs

2003; Kriventseva et al. 2003). Post-transcriptional

control allows a quicker response to environmental

stimuli compared with de novo transcription and in

some cases permits regulation of gene expression even

in the absence of transcription.

Splicing takes place in a large complex, called the

spliceosome, which comprises five small nuclear ribo-

nucleoprotein particles (snRNPs) and about two hun-

dred non-snRNP proteins (Zhou et al. 2002). Protein

factors essential for pre-mRNA processing in eukary-

otes have been identified and characterized primarily

through the study of nuclear extracts derived from

mammalian cells, Saccharomyces cerevisiae genetics

(Mount and Salz 2000), or through homology search-

ing. They include SR-rich RNA binding proteins

(Blencowe et al. 1999; Reddy 2004), protein kinases

(Kuroyanagi et al. 1998; Wang et al. 1998b; Savaldi-

Goldstein et al. 2003), phosphatases (Murray et al.

1999), RNA helicases (Tuteja 2000), peptidylprolyl cis–

trans-isomerases (Mortillaro and Berezney 1998;

Zhang et al. 2002; Lorkovic et al. 2004) and other

proteins with still unknown function. However, there

remain obstacles to the characterization of pre-mRNA

processing in plants. Despite attempts by several

groups to obtain active plant extracts for splicing in

vitro, a plant in vitro system is still not available, and

genetic approaches are more complex and less efficient

than in yeast or nematodes. Homology searching has

been used to identify RNA recognition motif (RRM)

and KH domain-containing RNA binding proteins in

Arabidopsis (Lorkovic and Barta 2002), but this ap-

proach may overlook factors with low sequence con-

servation, plant specific factors and factors connecting

pre-mRNA processing to other cellular processes. Gi-

ven these limitations, the yeast two-hybrid system

emerges as a useful additional tool for the character-

ization of RNA splicing, allowing the identification of

networks of protein–protein interactions in the

spliceosome, as has been demonstrated for yeast

(Fromont-Racine et al. 1997; Ben-Yehuda et al. 2000),

nematodes (Li et al. 2004), Drosophila (Stanyon and

Finley 2000) and plants (Golovkin and Reddy 1998,

1999; Lopato et al. 2002; Lorkovic et al. 2004). This

approach facilitates not only the cloning of cDNAs

encoding novel factors, but also the assignment of

possible function, inferred through the function of

interacting partners, and the grouping of proteins into

functional protein complexes.

Many splicing factors have been shown to be tissue

specific and are themselves developmentally regulated

both at the transcriptional and post-transcriptional

levels (Fu and Maniatis 1992; Screaton et al. 1995;

Hanamura et al. 1998; Lopato et al. 1999b, 2002; Ka-

lyna et al. 2003; Fang et al. 2004). Thus the spliceo-

some structure can be dynamic and might be specified

in different tissues or even groups of cells, regulating

organism development, by participation of tissue spe-

cific splicing factors. Several animal splicing factors

have been demonstrated to be involved in develop-

ment and, in particular, embryo development (for a

review, see Lopez 1998; Lalli et al. 2003), while gain-

and loss-of-function studies in plants indicate the cru-

cial role of alternative splicing in plant development

(Lopato et al. 1999b; Golovkin and Reddy 2003; Ka-

lyna et al. 2003; Savaldi-Goldstein et al. 2003).

Direct involvement in the early stages of embryo

development has recently been demonstrated for the

plant splicing factor AtRSZ33 (Kalyna et al. 2003).

AtRSZ33 is a nuclear localized plant specific RNA-

binding protein from Arabidopsis which is involved in

splicing, as demonstrated by its interaction with several

known splicing factors in the yeast two-hybrid system

and in pull-down assays (Lopato et al. 2002). Regula-

tion of alternative splicing of several genes has been

demonstrated in transgenic plants ectopically overex-

pressing AtRSZ33 (Kalyna et al. 2003). Moderate

levels of AtRSZ33 overexpression changed the cell

shape, and the plane of cell division in several differ-

entiating tissues became misorientated. The most

striking changes were found during early embryo

development, leading to either seed abortion or to the

638 Plant Mol Biol (2006) 62:637–653

123

appearance of monocotyledonous or twin plants (Ka-

lyna et al. 2003).

In monocotyledonous species, several examples of

alternative splicing regulation have been described

(Nash and Walbot 1992; Montag et al. 1995; Maga-

raggia et al. 1997; Locatelli et al. 2000; Halterman

et al. 2003). However, little is known about the

protein factors involved in pre-mRNA processing in

grasses and, particularly, about the role of such fac-

tors in grain development in crop plants. This

knowledge will provide us with the tools for manip-

ulating aspects of grain development for improved

yield or quality.

Here, the wheat (Triticum aestivum) homologue of

AtRSZ33 is described and shown to be expressed in the

embryo and in the mitotically active part of the syn-

cytial and cellularizing endosperm. This protein was

used as a starting bait to identify factors involved in

pre-mRNA processing in the early grain. A network of

their interactions are described for early wheat grain

using further yeast two-hybrid screens, in which preys

from previous screens were used sequentially as baits.

In addition to homologues of known factors involved in

pre-mRNA processing and transport in plants, animals

and yeast, cDNAs encoding several novel proteins

were identified.

Materials and methods

Plant material

Bread wheat (Triticum aestivum L. cv. Chinese Spring)

plants were grown in glasshouses with day tempera-

tures of 18–25�C and night temperatures of 18–21�C,

with a 13 h photoperiod. Spikes were labeled at

anthesis. Whole immature caryopses were harvested

daily at 0–6 days after pollination (DAP), and pooled

samples of liquid endosperm were collected at 3–7

DAP by pipette aspiration from the embryo sacs of

dissected micropylar tips of the grain.

mRNA Isolation and hybridization techniques

Total RNA was isolated from wheat samples using TRI

REAGENT (Molecular Research Centre, Inc., Cin-

cinnati, OH), and poly(A)+ RNA was then obtained

using an mRNA Direct Kit (Dynal, Norway), and used

for the preparation of MATCHMAKER Two-Hybrid

System 3 (Clontech, Palo Alta, CA) cDNA libraries.

In situ hybridization was performed according to

Weber et al. (1995) using [a-33P] dCTP-labeled cDNA

as a probe. To confirm the specificity of the in situ

hybridization several controls were performed. RNase

treatment before hybridization abolished any labeling

when using the same probe, indicating that the signal is

RNA specific. To check unspecific labeling we used as

a probe a 33P-labelled cDNA fragment encoding a

human methyltransferase gene with no known homol-

ogy to genes in higher plants (Rydberg et al. 1990) and

did not observe any labeling of our plant tissues with

this probe.

Plasmid construction and yeast two-hybrid

screening

The full-length coding region of the TaRSZ38 cDNA

was identified by constructing a contig of ESTs, and

was then amplified by PCR from the syncytial endo-

sperm cDNA library. The PCR fragment was subcl-

oned into the EcoRI–BamHI sites of the plasmid

pGBKT7 (Clontech) and used as a bait to screen the

above MATCHMAKER cDNA libraries. Two preys

of TaRSZ38, TaTra2 and TaTRN-SR were themselves

cloned into pGBKT7, as full-length and N-terminally

truncated sequences, respectively, either by PCR or by

direct subcloning of inserts from the prey construct

pGADT7-Rec. TaTra2 and TaTRN-SR were then used

as baits in subsequent two-hybrid screens of the same

cDNA libraries. Yeast transformation and screening

were performed essentially as described in the Yeast

MATCHMAKER System 3 manual. Putative positive

clones were verified by co-transformation with the bait

plasmid into yeast, and their sequences were analyzed

by HaeIII digestion, to identify redundant clones, and

direct sequencing. Full-length sequences of partial

clones identified in the two-hybrid screen, except

TaDRH1, were obtained using PCR of cDNA pre-

pared from wheat (cv. Chinese Spring) zygotic em-

bryos isolated at 5 DAP. The embryo-derived cDNA

had been synthesized and cloned into the phage vector

kTriplex2, as described in the SMARTTM cDNA Li-

brary Construction Kit (Clontech).

Quantitative PCR analysis

The primer pairs for TaRSZ38, TaPrp38 and

TaRSZBP1 were designed for the wheat variety Chi-

nese Spring using 3¢UTR sequences of cDNAs as a

template. The Q-PCR amplification was performed in

the RG 2000 Rotor-Gene Real Time Thermal Cycler

(Corbett Research, NSW, Australia) using QuantiTect

SYBR Green PCR reagent (Qiagen, VIC, Australia),

as described in Burton et al. (2004). The Rotor-Gene

V4.6 software (Corbett Research) was used to deter-

mine the optimal cycle threshold (CT) from dilution

Plant Mol Biol (2006) 62:637–653 639

123

series, and the mean expression level and standard

deviations for each set of four replicates for each

cDNA were calculated.

Protein expression

A PCR product containing the bacteriophage T7 pro-

moter, an N-terminal c-Myc epitope and the full-length

coding region of TaRSZ38 was used as a template to

express TaRSZ38 in vitro in the TnT Coupled Wheat

Germ Extract System Kit (Promega, NSW, Australia).

Five microliters of the reaction mix were analyzed by

SDS-PAGE, blotted onto nitrocellulose membrane,

and TaRSZ38 was immunodetected either with anti-

c-Myc monoclonal antibodies (Clontech) or with

antibodies against AtRSZ33 (Lopato et al. 2002). Pre-

immune preparations of antibodies were used as neg-

ative controls.

For expression in yeast (Saccharomyces cerevisiae),

the coding region of TaRSZ38 with an N-terminal c-

Myc epitope was subcloned into the pYES2.1 expres-

sional vector using the pYES2.1 TOPO TA Expression

Kit (Invitrogen, VIC, Australia). TaRSZ38 protein was

expressed in the EGY48 yeast strain and visualized by

western blots using an anti-c-Myc antibody (Clontech).

Fractionation of the syncytial endosperm and

immunodetection of RSZ proteins

Liquid endosperm from the caryopses of two spikes

was collected into 0.5 ml nuclei extraction buffer

(NEB) [25 mM Tris–HCl, pH 8.0, 0.44 M sucrose,

5 mM MgCl2, 2.5% Ficoll 400, 5% dextran, 0.5%

Triton X-100, 2 mM spermine, 2.5 mM dithiothreitol,

0.5 mM phenylmethylsulfonyl fluoride, protease

inhibitor cocktail (Sigma, concentration as recom-

mended in manual)]. The nuclei were isolated using a

protocol adapted from Chua et al. (2001). The liquid

part of the endosperm was centrifuged at 500g for

10 min at 4�C. The supernatant was collected and

used as a nuclei-free cytoplasmic fraction. The pellet

(partially purified nuclear fraction) was resuspended

in 20–100 ll NEB (without spermine) by gentle tap-

ping. The suspension of nuclei was loaded on a Per-

coll gradient (0.5 ml 80% and 0.5 ml 30% Percoll)

and centrifuged at 1,000g for 30 min at 4�C. The

intact nuclei were collected from the interface

between the 30 and 80% Percoll solutions. All frac-

tions were stained with 4¢,6-diamidino-2-phenylindole

and assessed by visualization under a fluorescence

phase microscope (Zeiss Axioskop; Carl Zeiss Pty.

Ltd., NSW, Australia) with the Zeiss number 2 filter

set (excitation filter 365 nm, barrier filter 420 nm) to

determine the fraction with the majority of nuclei and

minimum contamination.

The layer of nuclei was carefully removed and 10- to

20-fold diluted with NEB (without spermine). Nuclei

were pelleted by centrifugation at 1,500g at 4�C and

used for SDS-PAGE as a fraction of pure nuclei.

Proteins were loaded onto SDS-PAGE gels on an

equal protein basis, blotted onto nitrocellulose mem-

branes (Hybond C-extra; Amersham Biosciences,

NSW, Australia) and stained with Ponceau S. Wheat

RSZ proteins were immunodetected on western blots

using polyclonal antibodies raised against AtRSZ33

and AtRSZp22 (Lopato et al. 1999a, 2002). Pre-im-

mune antiserum was used as a negative control.

Sequence data from this article have been deposited

with the EMBL/GenBank data libraries under acces-

sion numbers DQ019624–DQ019639.

Results

Structural organization and expression of TaRSZ38

A tblastn computer search (Altschul et al. 1997) of the

GenBank expressed sequence tag database (dbEST),

using as a query the translated full-length cDNA of the

AtRSZ33 gene from Arabidopsis (Accession

AJ293801), returned several wheat ESTs with a high

level of identity to the RNA recognition motif (RRM)

and zinc finger (ZF) domains of AtRSZ33. The contigs

of the ESTs resulted in two cDNAs, both with open

reading frames encoding proteins of 333 amino acids

and a calculated molecular mass of 38 kDa, designated

TaRSZ38 and TaRSZ38a (Fig. 1A). The TaRSZ38 and

TaRSZ38a proteins have the same well-defined mod-

ular domains as AtRSZ33 (Fig. 1A, B). Alignment of

the deduced protein sequences of TaRSZ38 and

TaRSZ38a to AtRSZ33 and the related AtRSZ32

(Lopato et al. 2002) demonstrated a high level of

conservation in the RRM domain, the two CCHC-type

zinc fingers and the adjacent short RS-rich domain.

The identity across the RNA-binding domains is

slightly higher between TaRSZ38 and AtRSZ33 (68%

identity) than between TaRSZ38a and AtRSZ33 (66%

identity). In contrast, the SP domain of the wheat

proteins is longer and has no sequence similarity to the

SP domain of AtRSZ33. The level of conservation

between the two wheat proteins in the C-terminal part

is also low. No genes or ESTs were found in the

databases of Arabidopsis using the variable C-terminal

part of the wheat proteins. Therefore, TaRSZ38 has

the highest level of identity to AtRSZ33 of any known

or deduced wheat protein. TaRSZ38 is a plant specific

640 Plant Mol Biol (2006) 62:637–653

123

protein, as no clear orthologue of the TaRSZ38 or

AtRSZ33 proteins can be found in animals or yeast.

TaRSZ38 is expressed in mitotically active tissue

The expression of the gene encoding TaRSZ38 was

examined by real-time quantitative PCR in different

wheat tissues. The gene was expressed in all tissues,

though at varying levels. The highest level of expres-

sion was observed in flowers and at early stages of

grain development [0–5 days after pollination (DAP)].

At 5 DAP the highest level of expression was found in

the liquid part of the syncytial endosperm and in the

embryo and embryo surrounding tissue, with relatively

low levels of expression in the maternal tissue of the

grain (Fig. 2).

The spatial expression pattern of TaRSZ38 was

examined in wheat grain at 2, 4 and 8 DAP using

radioactive in situ hybridization. A cDNA fragment

containing sequences of the variable SP domain and the

3¢ untranslated region of TaRSZ38 was used as a probe.

At the 2 DAP stage, labeling covered all nucellar tis-

sues with the highest intensity in cell layers adjacent to

the endosperm cavity (Fig. 3A–C). Later, at 4 DAP, the

same low transcript levels are maintained in nucellar

tissues, but the labeling increased towards the interior

of the caryopsis and reached a maximum in the coe-

nocytic endosperm. In particular, intense labeling ap-

peared in the cytoplasm surrounding the multiple free

nuclei of the coenocyte, coinciding temporally with a

high rate of free nuclear divisions (Fig. 3D–F). In cell-

ularized endosperm (8 DAP), labeling revealed high

1 121AtRSZ32 (1) MPRYDDR---YGNTRLYVGRLSSRTRTRDLERLFSRYGRVRDVDMKRDYAFVEFSDPRDADDARYYLDGRDFDGSRITVEASRGAPRGS-------------RDNGSRGPPPGSGRCFNCGAtRSZ33 (1) MPRYDDR---YGNTRLYVGRLSSRTRTRDLERLFSRYGRVRDVDMKRDYAFVEFGDPRDADDARHYLDGRDFDGSRITVEFSRGAPRGS-------------RDFDSRGPPPGAGRCFNCGTaRSZ38 (1) MPRYDDR---YGNARLYVGRLSSRTRSRDLEYLFSKYGRIREVELKRDYAFIEYSDPRDADEARYNLDGRDVDGSRIIVEFAKGVPRGSGGS--------REREYVGRGPPPGTGRCFNCG

TaRSZ38a (1) MPRYDERDRYGGNTRLYVGRLPSRTRTRDLEDLFGRYGRVRHVDMKHEFAFVEFSDPRDADEARYNLDGRDFDGSRMIVEFAKGVPRGQGGGGDRDRGRGGDREYMGRGPPPGSGRCFNCG

122 242AtRSZ32 (106) VDGHWARDCTAGDWKNKCYRCGERGHIERNCKNSPSPKKARQGGSYSRSPVKSRSPRRRRSPSRSRSYSRGRSYSRSRSPVRR--EKSVEDRSRSPKA---MERSVSPKGR---DQSLSPDAtRSZ33 (106) VDGHWARDCTAGDWKNKCYRCGERGHIERNCKNQP--KKLRRSGSYSRSPVRSRSPRRRRSPSRS--LSRSGSYSRSRSPVRRR-ERSVEERSRSPKR---MDDSLSPRAR---DRSPVLDTaRSZ38 (111) IDGHWARDCKAGDWKNKCYRCGERGHIERNCQNSP--RSLRRERSYSRSPSPRRGRARSRSYSRSRSYSRSRSRSYSESPRGRR-TERDERRSRSISYSRSPRRSLSPGGKE-MDRSPTPD

TaRSZ38a (122) IDGHWARDCKAGDWKNRCYRCGDSGHIERDCQNSP--KNLKRGKSYSRSPSPRRGRVRDRSYSRS----RSRSYSRSVSPRRDERRSRSPRDSRSPRR--SPRDSRSPRRSPRDSRSPRRS

243 362AtRSZ32 (219) R------------KVIDASP---------------KRGSDYDG-SPKENGNGR----------NSASPIVGGGESPV-----------GLNGQDRSPIDDEAELSRPSPKG-SESP---- AtRSZ33 (216) DEGSP--------KIIDGSPP-------PSPKLQKEVGSDRDGGSPQDNGRN-----------SVVSPVVGAGG--------------DSSKEDRSPVDDDYEPNRTSPRG-SESP---- TaRSZ38 (228) RSRSPRRSISPVAKDNGDSPRGRETSRSPSDGYRSPVANGRSPRSPVNNGSPSP------TRDNRASPSLRGNNGSPSPKGNGN----GGSPSPRGNGDDDGRRGSGSPRGRSVSP----

TaRSZ38a (235) PRDS---------RSPMKSPR----------GSRSPMRSPRDSRSPRRSASPAKGRARSPTPPGSRSPAPRENSRSPMKADSPNNMSPAANGRSPSPRDGEDNGNHRAPSG-SASPGGG-

(A)

Binding site for the rest of identified proteins

ZnF RS SP

AtRSZ33

TaRSZ38

Binding sites forSCL proteins

Binding sites for TaTra2

RRM

ZnF RS SPRRM

(B)kDa

250 -

98 -64 -

50 -

36 -

30 -

16 -

anti c-Myc PI anti RSZ33

(C)

Fig. 1 The structure of RSZ proteins from wheat and Arabid-opsis. (A) Alignment of the protein sequences of TaRSZ38,TaRSZ38a, AtRSZ32 and AtRSZ33. Protein domains areunderlined with different color bars, identified in panel (B).(B) Domain structures of AtRSZ33 and TaRSZ38: RRM—RNArecognition motif; ZnF—two CCHC-type RNA binding zincfingers; RS—SR-rich protein binding domain; SP—less con-

served sequence with multiple SP dipeptides surrounded byother amino acids. Protein binding domains are indicated. (C)Immunodetection of TaRSZ38 expressed in wheat germ extractswith an N-terminal c-Myc epitope. The antibodies used wereagainst the c-Myc epitope (anti c-Myc), preimmune antibodies(PI), or antibodies against AtRSZ33 truncated in the SP domain(anti RSZ33)

Plant Mol Biol (2006) 62:637–653 641

123

expression of TaRSZ38 at the periphery of the endo-

sperm, in mitotically active cells with dense cytoplasm

and a low content of starch grains. The level of

expression decreases toward the inner region of the

endosperm, where the major storage tissue of the grain

at this time is characterized by cells of different size

with large vacuoles and large starch granules (Fig. 3G–

I). The labeling pattern described here therefore im-

plies preferential expression of TaRSZ38 in mitotically

active tissues of the developing caryopsis.

Immunodetection of TaRSZ38 in the nuclear

fraction of the syncytial endosperm

The large syncytial endosperm of wheat provided a

unique opportunity to purify intact nuclei and nuclei-

free cytoplasmic fractions from a single multinucleate

cell. The high level of sequence similarity between

TaRSZ38 and AtRSZ33 suggested also that the wheat

protein might be recognized by antibodies raised

against AtRSZ33 from Arabidopsis (Lopato et al.

2002). To test this, TaRSZ38 was expressed with an

N-terminal c-Myc epitope in wheat germ extracts, and

found to cross-react with the anti c-Myc and anti-

AtRSZ33 antibodies (Fig. 1C). Analyses of the

cytoplasmic and nuclear fractions of the syncytial

endosperm using antibodies against AtRSZ33 revealed

one band in the nuclei-free cytoplasm and several

protein bands in the nuclear fraction with a major

protein band at about 64 kDa (Fig. 4). Expression of

recombinant TaRSZ38 in eukaryotic yeast cells, which

contain homologues of specific SR protein kinases

(Siebel et al. 1999), resulted in a single protein band of

the same size (Fig. 4). Therefore it appears that the

64 kDa protein band represented a form of TaRSZ38

that had been modified post-translationally, running

more slowly in SDS-PAGE than the unmodified form

(deduced molecular weight of 38 kDa). A similar shift

TaPrp38

0

5000

10000

15000

20000

Co

pie

s/m

icro

gra

m o

f R

NA

0

15000

30000

45000

60000

Inflo

resc

ence

Flowers

, meio

sis

Flowers

1 d pre

-pol

Grain

0-1 D

AP

Grain

2-3 D

AP

Grain

4-5

DAP

Grain

6-7 D

AP

Grain

8-1

0 DAP

Grain

11-15

DAP

Grain

16-20

DAP

Embryo (e

nrich

ed) 5 D

AP

Endo

sper

m (liqu

id) 5

DAP

Pericarp

5 D

AP

Roots (s

eedling)

Shoots

(seed

ling)

Wou

nded

LeafSte

m

Cop

ies/

mic

rogr

am o

f RN

A

TaRSZBP1

Flowers Developing grain Grain fractions Vegetative tissue

0

5000

10000

15000

20000

25000

Co

pie

s/m

icro

gra

m o

f R

NA

TaRSZ38

Fig. 2 Expression of TaRSZ38, TaPrp38 and TaRSZBP1 at different stages of flower and grain development, in grain fractions andvegetative tissue, as determined by quantitative real-time PCR. Data are normalized against the input amount of RNA

642 Plant Mol Biol (2006) 62:637–653

123

in apparent size has been observed for other SR pro-

teins following phosphorylation (Lopato et al. 1999b).

The same endosperm fractions were examined with

a second antibody raised against another Arabidopsis

zinc finger containing protein, AtRSZ22 (Lopato et al.

1999a), which was later identified in the same protein

complex as AtRSZ33 (Lopato et al. 2002). A single

protein band with an approximate molecular weight of

37 kDa was identified in the nuclear but not in the

cytoplasmic fraction, representing the post-transla-

tionally modified form of a RSZ22-like protein(s) from

wheat.

Interacting partners of TaRSZ38 are involved in

pre-mRNA processing

Proteins which function in a complex with TaRSZ38 in

early wheat grain were sought using yeast two-hybrid

screens of cDNA libraries prepared from whole,

immature wheat grains (0–6 DAP) or the syncytial

endosperm (3–7 DAP). About 100 clones that

activated the reporters only in the presence of the

TaRSZ38-fusion bait were isolated from the wheat

grain cDNA library and characterized by PCR and

restriction digestion with HaeIII. Clones representing

seven different proteins were identified and sequenced.

Six clones encoded proteins with structural and

sequence homology to known animal, yeast or plant

factors involved in pre-mRNA processing (for align-

ments of all wheat proteins discussed herein with

known proteins from other species, see Supplemental

Data I). The remaining clone encoded an unknown

protein, designated TaRSZBP1. The proteins inter-

acting with TaRSZ38 as well as the names of their

plant, yeast and animal homologues are listed in

Table 1, and their interactions are displayed in Fig. 5.

Three groups of clones were isolated from the wheat

endosperm library. They represented three proteins

(TaPRP38, TaLuc7 and TaU1-70K) that were also

isolated from the whole grain library, except the

abundance and length of clones differed (see Supple-

mental Data II for data relating to the abundance and

length of the prey clones analyzed from each of the two

libraries). The most abundant from the endosperm li-

brary (90% of all clones) was TaPRP38, whereas only

3% of clones isolated from the grain library encoded

Fig. 3 Spatial expression patterns of TaRSZ38 within immaturewheat caryopses at 2 DAP (A–C), 4 DAP (D–F), and 13 DAP(G–I). Panels A (transverse), D and G (longitudinal) demon-strate tissue structure and the location of micrograph fields insubsequent frames. Panels B, E and H are dark-field images inwhich TaRSZ38 mRNA appears as a white emulsion. F and I arethe corresponding light-field images. Panel C shows a highermagnification of two recently divided nuclei, stained blue, in the

layer of free nuclei surrounding the central vacuole; thehybridization signal is shown in black. Iodine staining showsstarch granule accumulation in the cellularized endosperm at 13DAP (I). Pericarp (p), nucellus (n), filial tissues (f), vacuole (v),coenocytic vacuole (c), endosperm (e), pericarp ventral (pv),nucellar projection (np), inner and peripheral regions ofendosperm (ie and pe, respectively)

Plant Mol Biol (2006) 62:637–653 643

123

this protein. This observation was corroborated by

quantitative real-time PCR analysis, which showed that

TaPrp38 transcript is 13- and 20-fold more abundant in

the liquid endosperm than in the pericarp and embryo,

respectively (Fig. 2). Clones encoding the other four

proteins identified in the wheat grain cDNA library

(TaTra2, TaU2AF65, TaTRN-SR, TaRSZBP1) were

not found in the endosperm cDNA library. This could

reflect the fact that these proteins are not expressed in

the endosperm, although real-time PCR showed

TaRSZBP1 transcript levels, at least, to be relatively

abundant in liquid endosperm (Fig. 2). An alternative

explanation, then, is that the apparent abundance of

clones in the endosperm two-hybrid library was af-

fected by the lower average size of inserts in this li-

brary. The full-length clones encoding TaTRN-SR,

TaLuc7, TaPrp38 and TaU2AF35 were isolated using

PCR from a cDNA library prepared from zygotic

embryos at 5 DAP, and a full-length clone of TaTra2

was amplified from wheat grain cDNA.

Prey proteins interact with the TaRSZ38 RS or SP

domains

Deletions of the coding region of TaRSZ38, to produce a

series of truncated proteins, were cloned into the bait

plasmid and used in the yeast two hybrid assay to identify

the segments which interact with prey proteins. The

relative strengths of protein-protein interactions were

determined by testing the ability of the yeast to grow on

His(–) Ade(–) selective medium (Table 2). All prey

proteins strongly interacted with a full-length fragment

of TaRSZ38 minus the SP domain (Fig. 1B). Truncated

protein, containing the SP domain only, showed weak

interaction with TaPrp38 and TaTRN-SR, but no

interaction with other proteins. However, absence of the

SP domain does not lead to a noticeable change in the

interaction of TaRSZ38 with TaPrp38 and TaTRN-SR.

The RS domain of TaRSZ38 is sufficient for the strong

interaction with all proteins except TaTra2, for which

the strong interaction requires a short segment between

the RRM and zinc fingers (Fig. 1B). The length of clones

and domain structure of prey proteins summarized on

Fig. 6 provide partial mapping of protein segments of

preys that interact with TaRSZ38.

Tosummarize, theRSdomainofTaRSZ38issufficient

for the protein–protein interaction with each of seven

proteins, but the strength or specificity of the interaction

is modulated by other segments of the protein.

TaTra2, like TaRSZ38, interacts with an SR

transportin and homologues of Arabidopsis splicing

factors

In order to build up a network of splicing factors and

associated proteins in monocots, sequential two-hybrid

Fig. 4 RSZ proteins are enriched in the nuclear fraction ofwheat syncitial endosperm. (A) Purification quality control ofcrude (1) and Percoll-gradient purified (2) nuclear fractions ofthe liquid syncytial endosperm of wheat stained with DAPI. (B)Immunodetection of RSZ proteins in wheat endosperm frac-

tions. (C) Immunodetection of recombinant TaRSZ38 expressedin yeast. Lane 1—cytoplasmic fraction; 2—crude nuclear frac-tion; 3—nuclear fraction after Percoll-gradient purification; 4–7—protein expression in yeast: 4–0 h; 5–4 h; 6–8 h; 7–24 h afterinduction

644 Plant Mol Biol (2006) 62:637–653

123

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Plant Mol Biol (2006) 62:637–653 645

123

screens were performed using successive preys as baits.

TaTra2, from whole wheat grain, was first identified as

a prey protein for the TaRSZ38 bait. Tra2 factors in

animals play an important role in embryo development

(Ryner and Baker 1991), generally acting to activate

alternative splice sites that would not otherwise be

used. The cloned wheat protein displayed a high level

of structural similarity to animal Tra2 proteins with

49% sequence identity in the RRM domain, suggesting

that the wheat protein may be a functional homologue

of the animal proteins. Full-length TaTra2 was used as

bait in a yeast two-hybrid screen of the wheat grain

library, and clones encoding five proteins were identi-

fied (Fig. 5). Four groups of clones encode two pairs of

close homologues of the Arabidopsis splicing factors

AtSRp30 and AtRSZ22. The fifth group of clones

encodes the same SR transportin, TaTRN-SR, that was

found when using TaRSZ38 as a bait. Thus, taken to-

gether, the plant homologue of Tra2 factor interacts

with proteins that have structural and functional simi-

larity to animal splicing factors SF2/ASF and 9G8,

which themselves physically and/or functionally inter-

act with Tra2 (Amrein et al. 1994). TaTra2 also ap-

pears to be delivered into the nucleus by the same

transportin as TaRSZ38. The proteins interacting with

TaTra2 as well as the names of their plant, yeast and

animal homologues are listed in Table 1.

TaTRN-SR transportin interacts with further RNA-

binding proteins

TaTRN-SR is a plant transportin with the highest level

of protein sequence identity to the yeast Mtr10 and

animal TRN-SR transportins. These proteins are

known to directly bind splicing factors and transport

them from the cytoplasm to the nucleus. It was dem-

onstrated above that two wheat splicing factors,

TaRSZ38 and TaTra2, strongly interact with TaTRN-

SR in the yeast two-hybrid assay. Further screening of

the wheat grain cDNA library, using a 1.8 kb fragment

of TaTRN-SR without the N-terminal binding site for

the ‘‘cargo unloading’’ protein Ran-GTP, revealed

interactions of TaTRN-SR with further RNA-binding

proteins (Fig. 5). The most abundant group of clones

(more than 80% of analyzed clones) encoded partial

and full-length clones homologous to animal Y14

(Tsunagi), a factor involved in mRNA export and

which has been implicated in the cytoplasmic nonsense

mediated decay (NMD) complex (Le Hir et al. 2001)

and which also plays an important role in the devel-

opment of the embryo in Drosophila (Mohr et al.

2001). The high abundance of TaY14 clones in the li-

brary unfortunately made the search for other factors

time and labor consuming. Nevertheless, several clones

were found, encoding wheat homologues of known

animal factors involved in splicing, mRNA export and

transcription (Table 1), implicating TaTRN-SR in the

nuclear import of pre-mRNA processing and export

factors. In addition, a collection of partial sequences of

several unidentified proteins with RD-rich sequences

or hydrophobic signal peptides was isolated (data not

shown).

Discussion

Post-transcriptional processing of pre-mRNA signifi-

cantly affects the diversity of the end products of gene

expression. Considerable data have appeared over the

last decade about the mechanisms of splicing and the

factors involved in constitutive and alternative splicing

in animals and yeast. However, the data on splicing in

plants remain sparse and what little is known is based

largely on the model species Arabidopsis. Our interest

is grain development in wheat, a crop plant with a very

large, unsequenced genome. It is expected that post-

transcriptional regulation is important in embryo

development, as shown for animals (Lopez 1998; Groth

Table 2 Mapping of segments of TaRSZ38 which are involved in the interaction with proteins identified in the Y2H screen

Bait Prey

pGADT7 TaLuc7 TaPrp38 TaTRN-SR TaU1-70k TaU2AF65 TaRSZBP1 TaTra2

pGBKT7 – – – – – – – –TaRSZ38 1–333 aa – +++ +++ +++ +++ +++ +++ +++DRRM 82–333 aa – +++ +++ +++ +++ +++ +++ +++DRRM and RS1 99–333 aa – +++ +++ +++ +++ +++ +++ +––DRRM, RS1 and 2ZnF 147–333 aa – +++ +++ +++ +++ +++ +++ +––SP only 180-333 aa – – +–– ++– – – – –DSP 1–208 aa – +++ +++ +++ +++ +++ +++ +++

D, deleted region; RRM, RNA recognition motif; RS1, small arginine–serine rich region between RRM and first zinc finger; 2ZnF,CCHC-type zinc finger; SP, serine–proline rich region

646 Plant Mol Biol (2006) 62:637–653

123

AD-TaLuc7

AD-TaU1-70K

pGADT7

AD-TaPrp38

AD-TaTra2

AD-TaTRN-SR

AD-TaU2AF65

AD-TaRSZBP1

pGBKT7 BD-TaRSZ38 BD-TaRSZ38pGBKT7

-Leu, -Trp -Leu, -Trp, -His, -Ade, + X-GAL

pGADT7

AD-TaSRp30

AD-TaSRp30a

AD-TaRSZ22

AD-TaRSZ22a

AD-TaTRN-SR

pGBKT7 BD-TaTra2 pGBKT7 BD-TaTra2

pGADT7

AD-DRH1

AD-U2AF35

AD-TaY14

AD-TaU2AF65

pGBKT7 BD-TaTRN-SR pGBKT7BD-TaTRN-SR

TaRSZBP1

TaU2AF65

TaTra2

TaPrp38

TaLuc7

TaU1-70K

TaTRN-SR

TaSRp30

TaRSZ22

TaU2AF35TaRSZ38

TaSRp30aTaRSZ22a

TaDRH1

TaY14

Unknown proteinswith SD-rich sequences

TaU2AF65a

Data obtained for wheat grain

Published data for other organisms

(A)

(B)

(C)

(D)

Plant Mol Biol (2006) 62:637–653 647

123

and Lardelli 2002; Lalli et al. 2003) and Arabidopsis

(Kalyna et al. 2003), and exerts some control over such

factors as embryo size and composition. Equally, an

understanding of the specific features of splicing reg-

ulation in the developing endosperm may provide tools

for manipulating endosperm size, composition or

morphology, and consequently grain yield and quality.

As a starting bait in yeast two-hybrid screens of

cDNA libraries prepared from early wheat grain (0–6

DAP) and the liquid part of the syncytial endosperm

(3–7 DAP), the wheat homologue of AtRSZ33 was

chosen as this is the only plant splicing factor with a

demonstrated role in early seed development (Kalyna

et al. 2003). This protein is expressed in different plant

tissues, but only in cells undergoing active division and

elongation. Overexpression of AtRSZ33 in transgenic

Arabidopsis resulted in several pronounced changes in

plant development (Kalyna et al. 2003). The most

interesting changes took place in the early stages of

embryo development, with the appearance of either

twin or monocotyledonous plants after germination.

The wheat homologue of AtRSZ33 was expressed in all

tissues examined, with the highest level of expression

in developing grain. These data correlate with expres-

sion data obtained earlier for AtRSZ33 using pro-

moter:GUS constructs (Lopato et al. 2002; Lorkovic

and Barta 2002; Kalyna et al. 2003). AtRSZ33 is

expressed in restricted groups of cells in different

organs of Arabidopsis including seeds. The large size of

the wheat grain relative to Arabidopsis was exploited

to demonstrate that TaRSZ38 is expressed mainly in

cells which are undergoing intense mitotic division.

The highest expression was found in the syncytium

around the dividing free nuclei. Expression of

TaRSZ38 in the dividing nuclei of the coenocyte was

further supported by immunodetection of the

TaRSZ38 protein in the nuclear fraction purified from

syncytial endosperm. These data suggest that the role

of TaRSZ38 is not restricted to the regulation of em-

bryo development, but might be extended to endo-

sperm development. Although it was demonstrated

earlier that AtRSZ33 is expressed in Arabidopsis

endosperm (Kalyna et al. 2003), changes in endosperm

in transgenic plants overexpressing AtRSZ33 were not

studied.

A previous two-hybrid screen with AtRSZ33 from

Arabidopsis did not include the SP domain in the bait

construct, and clones from organs such as flowers and

siliques were under-represented in the prey library

(Lopato et al. 2002). In contrast, the full-length

TaRSZ38 could be expressed successfully in yeast,

providing an ideal bait. Two cDNA libraries were

screened, prepared from the whole grain and liquid

coenocyte at overlapping stages of development. Six

from seven identified proteins were either known

splicing factors or splicing related proteins. In a study

of this size, the usual method for confirming yeast two-

hybrid data, pull-down assays, is extremely difficult to

achieve. Protein phosphorylation is a likely pre-

requisite for correct protein interactions, particularly

involving RS domains, precluding the use of re-

combinant proteins expressed in bacteria, while pull-

down assays using plant material would require the

generation of antibodies specific to each protein. In the

circumstances, we have corroborated the observed

yeast two-hybrid data by comparison to protein inter-

action databases that include homologues of the cloned

wheat genes.

No members of the SC35-like (SCL) family of pro-

teins, identified in a previous study (Lopato et al.

2002), were found in these screens. One prey protein

(TaTra2) resembles SCL proteins, having a single

RRM domain in the central part of the protein (Fig. 6),

but the wheat protein is in fact most closely related to

animal Tra2 splicing factor (49% identity in the RRM

domain). Animal Tra2 factors are important for

development, blocking splice sites in specific

pre-mRNAs during the early stages of Drosophila

embryo development (Ryner and Baker 1991) and

likely participating in the control of human cell specific

splicing patterns (Tacke et al. 1998). TaTra2 interacts

in the grain with other splicing factors and may have a

similar role in regulating splicing events during devel-

opment. Despite the fact that TaTra2 and SCL proteins

are structurally distinct, mapping of protein–protein

interactions demonstrated that they interact with the

same segments of RSZ proteins.

Homologues of two further proteins identified in the

screen with RSZ33 from Arabidopsis, AtSRp34 and

AtRSZ22, were not found in the screen with wheat

TaRSZ38. Surprisingly, several homologues of each of

Fig. 5 Protein–protein interaction data. (A–C) reconstitution oftwo-hybrid interactions found in yeast two-hybrid screening.Yeast cells were co-transformed either with prey plasmidsisolated in a yeast two-hybrid screening (indicated at the leftside) and empty bait vector (pGBKT7), or with the preyplasmids and the bait plasmid used in the respective screens(A, BD-TaRSZ38; B, BD-TaTra2; and C, BD-TaTRN-SR).Yeast cells were maintained on selective media without leucineand tryptophan (-Leu, -Trp) or without leucine, tryptophan,histidine and adenine, but with X-GAL to test for galactosidaseactivity (-Leu, -Trp, -His, -Ade, + X-GAL). (D) Schematicrepresentation of protein–protein interaction data obtained fromthe yeast two-hybrid screens and partially confirmed by datapublished for other organisms. AD, activation domain; BD,binding domain

m648 Plant Mol Biol (2006) 62:637–653

123

these proteins were found in a subsequent two-hybrid

screen with TaTra2 as bait. This supports the idea that

in wheat and Arabidopsis small 9G8-like RSZ proteins

and the more evolutionary conserved SF2/ASF-like

SRp proteins function in the same complex together

with either Tra2-like and/or SCL proteins. Interaction

of the Drosophila splicing regulator Tra2 with the SF2/

ASF splicing factor has been demonstrated in vitro and

in the yeast two-hybrid assay (Wu and Maniatis 1993;

Amrein et al. 1994). It was also demonstrated in

human cell extracts that Tra2 recognizes purine-rich

enhancer elements and recruits to these elements 9G8

and SF2/ASF proteins (Lynch and Maniatis 1996).

TaRSZ38 appears to be a plant specific member of a

similar protein complex in the plant spliceosome,

which might be responsible for plant specific regulation

of constitutive and/or alternative splicing.

It was also found that TaRSZ38 can directly and

strongly interact with TaU1-70K and TaLuc7 proteins.

U1-70K from metazoans has been shown to interact

with SR-splicing factors SC35, ASF/SF2 and Sip1, and is

involved in both basic and alternative splicing (Wu and

RDE

TaU2AF65

RS RSTaTra2 P

PRP38 RDH RSKTaPrp38

LUC7 RDRSTaLuc7

TaU1-70K

RDH

TaTRN-SR

TaRSZBP1

RSTaSRp30

7X(-GRRRSSRREDHSSDG-)

RSTaRSZ22 ZFRRM

RRM

RRM ψRRM

RRM

RRM RRM RRM

RSDETaU2AF35 ψRRM

RSPTaY14 RRM

C2H2C3H

Ran-BD RS-BD

RG RG

C3H

TaDRH1 RGRSHELICDEAD

Fig. 6 Domain structure of the proteins identified in yeast two-hybrid screens: RRM, RNA recognition motif; YRRM, stronglydegenerated version of RRM; TD, transmembrane domain;Kinase domain, S/T kinase active center; Ran-BD, Ran-GTP-binding domain; RS-BD, SR-protein binding domain; ZF, CCHC-type zinc fingers; C3H and C2H2, CCCH and CCHH zinc fingers;HELIC, RNA helicase domain; DEAD, signature sequence ofDEAD box RNA helicases; LUC7 and PRP38, conserved

domains within the families of proteins with unknown function;P, proline-rich sequence usually with proline stretches; RS, RSK,RDRS, RDE, RSP and RGRS, usually protein binding domains(sequences) enriched in particular amino acids with repeateddipeptides containing arginines; 7X(-GRRRSSRREDHSSDG-),seven near perfect repeats of 15 amino acids. The length ofproteins deduced from clones obtained in the yeast two-hybridscreen is indicated with arrows

Plant Mol Biol (2006) 62:637–653 649

123

Maniatis 1993; Kohtz et al. 1994; Manley and Tacke

1996). The homologue of U1-70K from Arabidopsis can

interact in vivo and in vitro with small RSZ22-like

proteins and SC35-like proteins (Golovkin and Reddy

1998). Luc7p is a component of the yeast U1 small

nuclear ribonucleoprotein particle (snRNP) and is

essential for the formation of the spliceosome com-

mitment complex in vitro (Fortes et al. 1999). In the

absence of Luc7p the protein composition of the U1

snRNP is changed, splicing efficiency is reduced and 5¢splice site selection is altered. Isolation of plant ho-

mologues of Luc7p had not been reported previously.

Interaction of TaRSZ38 with two proteins bound to

the U1 snRNP suggests involvement of this protein in

the early steps of spliceosome assembly and in the

recognition of the 5¢ splice site. However, interaction

of TaRSZ38 with another protein, designated TaPrp38,

suggests that TaRSZ38 participates in splicing at least

until the end of cleavage of the first exon. Prp38p is an

essential splicing factor in yeast, as a mutation in the

PRP38 gene is lethal. In the temperature-sensitive

mutant prp38, high temperature prevents the first

cleavage-ligation event in excision of introns from

mRNA precursors (Blanton et al. 1992). In the ab-

sence of Prp38p activity, spliceosomes form but they

arrest in a catalytically impaired state (Xie et al. 1998).

Nothing is known about homologues of Prp38p in

multicellular organisms, although database searches

for nematodes indicate embryo specific expression of a

related protein, for which RNAi silencing caused early

embryo defects, reabsorbed polar bodies, and embry-

onic lethality (see annotation to the gene product

Accession NP_505762). TaPrp38 is also expressed in

wheat embryos, as we could amplify the full-length

cDNA using a library prepared from zygotic embryos

isolated at 5 DAP. An important role for TaPrp38 in

endosperm development is supported by the relative

abundance of TaPrp38 among clones isolated in the

yeast two-hybrid screen from the wheat endosperm

cDNA library (90% of all clones) and 13- to 20-fold

higher abundance of TaPrp38 mRNA in the liquid

endosperm in comparison with the rest of the grain.

An additional involvement of TaRSZ38 in 3¢ splice

site regulation cannot be excluded, since it interacts

U2AF35

A

E1 E2Branchpoint

Polypyrimidine tract

5’ splicesite

3’ splicesite

A UUUUU

U1 snRNP U2 snRNPTra2U2AF35

SRpU1-70K

Luc7

Capping complex recruitment

Enhancer

U5 snRNP

U6 snRNP

U4 snRNP

Prp38

UUUUUA

U1 snRNP

U4 snRNP

U2 snRNPU6 snRNP

U5 snRNPPrp38U6/U4 unwinding

UUUUU

UUUUUA

Pre-mRNA

Pre-spliceosome

Spliceosome

Spliced mRNA Lariat intron

+

U2 snRNP recruitment andalternative splice site selection

U1 snRNPrecruitment andalternative splice site selection

RSZ22

Y14

Luc7

U1-70K

Tra2

Prp38

TaLuc7

TaU1-70K

TaU2AF65

TaTra2

TaPrp38

U2AF35

U2AF65

TaU2AF35

SRp TaSF2/ASF-like(TaSRp30, TaSRp30a)

RSZ22 TaRSZ22-like(TaRSZ22, TaRSZ22a)

Y14 TaY14

Proteins isolated with TaRSZ38

Proteins isolated with TaTra2 andTaTRN-SR

mRNA export

U2AF35

Fig. 7 Schematic representation of a typical splicing reaction, showing the deduced positions and functions of proteins identified in theyeast two-hybrid screen

650 Plant Mol Biol (2006) 62:637–653

123

with U2AF65, the large subunit of the U2 auxiliary

factor. Animal U2AF65 recruits U2 snRNPs by inter-

action with U2 snRNP components (Gozani et al.

1998; Wang et al. 1998a). The polypyrimidine tract,

which is located on the intron sequence between the

branch point and 3¢ splice site, is recognized by the

large subunit of U2AF (Zamore et al. 1992).

In the two screens with TaRSZ38 and TaTra2 as

baits the same partial clones were isolated that encode

a protein with a high identity to Mtr10 from yeast and

animal TRN-SR. It has been demonstrated that nu-

clear import of splicing factors with SR domains is

mediated by a specific import receptor, termed trans-

portin-SR (TRN-SR). TRN-SR is a member of the

importin b family and it binds directly, without the help

of any a subunit, to nuclear localization signals (NLSs)

within SR domains (Kataoka et al. 1999; Lai et al.

2000). Using a partial sequence of TaTRN-SR as bait it

was found that this protein identifies several other

putative splicing factors and factors involved in mRNA

export. These include cDNA clones encoding both

subunits of U2AF splicing factor, an RNA-helicase, a

homologue of the animal Y14 protein and several

partial clones encoding unidentified proteins contain-

ing SD-rich sequences.

Mtr10p is the closest yeast homologue to TRN-SR.

This is a nuclear import receptor specialized in the

transport of RNA-binding proteins. A mutation in

MTR10 also causes accumulation of poly(A)+ RNA in

the nucleus (Kadowaki et al. 1994) and the question

arises if Mtr10p can be either directly or indirectly (by

delivery of export factors to the nucleus) involved in

mRNA export. The participation also of TaTRN-SR in

mRNA export cannot be excluded, as it interacted with

both Y14 and subunits of U2AF, known to be involved

in mRNA export (Le Hir et al. 2001; Zolotukhin et al.

2002; Singh and Lykke-Andersen, 2003; Blanchette

et al. 2004) and the cytoplasmic nonsense mediated

decay complex (Gatfield et al. 2003; Gehring et al.

2003).

This study has identified a number of proteins with

putative roles in RNA splicing during early grain

development in wheat. Figure 7 shows a schematic

representation of a typical splicing reaction, showing

the deduced positions and functions of the wheat

proteins identified in the yeast two-hybrid experiments.

The developing wheat grain is an interesting model for

this type of study. The transition from liquid to cellu-

larized endosperm is a key process in grain develop-

ment and modification of this may lead to alterations in

grain shape, size and composition. It seems probable

that RNA splicing is one component of the regulatory

machinery used for this transition. The size of the

wheat grain makes the analysis of endosperm devel-

opment far simpler than for Arabidopsis and, impor-

tantly, the uncellularized, liquid endosperm or

syncytium can be isolated and used as a source for

components of the splicing apparatus. Ultimately the

liquid endosperm may allow the development of an in

vitro splicing assay.

Acknowledgments We thank Ursula Langridge for assistancewith growing plants in the glasshouse. This work was supportedby the Australian Grain Research and Development Corpora-tion (grant no. UA00083 to P.L.).

References

Abler ML, Green PJ (1996) Control of mRNA stability in higherplants. Plant Mol Biol 32:63–78

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z,Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search pro-grams. Nucleic Acids Res 25:3389–3402

Amrein H, Hedley ML, Maniatis T (1994) The role of specificprotein-RNA and protein–protein interactions in positiveand negative control of pre-messenger-RNA splicing bytransformer-2. Cell 76:735–746

Barrass JD, Beggs JD (2003) Splicing goes global. Trends Genet19:295–298

Ben-Yehuda S, Dix I, Russell CS, McGarvey M, Beggs JD,Kupiec M (2000) Genetic and physical interactions betweenfactors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae. Genetics156:1503–1517

Blanchette M, Labourier E, Green RE, Brenner SE, Rio DC(2004) Genome-wide analysis reveals an unexpected func-tion for the Drosophila splicing factor U2AF(50) in thenuclear export of intronless mRNAs. Mol Cell 14:775–786

Blanton S, Srinivasan A, Rymond BC (1992) PRP38 encodes ayeast protein required for pre-mRNA splicing and mainte-nance of stable U6 small nuclear RNA levels. Mol Cell Biol12:3939–3947

Blencowe BJ, Bowman JAL, McCracken S, Rosonina E (1999)SR-related proteins and the processing of messenger RNAprecursors. Biochem Cell Biol 77:277–291

Burton RA, Shirley NJ, King BJ, Harvey AJ, Fincher GB (2004)The CesA gene family of barley. Quantitative analysis oftranscripts reveals two groups of co-expressed genes. PlantPhysiol 134:224–236

Chua YL, Brown AP, Gray JC (2001) Targeted histone acety-lation and altered nuclease accessibility over short regionsof the pea plastocyanin gene. Plant Cell 13:599–612

Domon C, Lorkovic ZJ, Valcarcel J, Filipowicz W (1998) Mul-tiple forms of the U2 small nuclear ribonucleoprotein aux-iliary factor U2AF subunits expressed in higher plants. JBiol Chem 273:34603–34610

Ewing B, Green P (2000) Analysis of expressed sequence tagsindicates 35,000 human genes. Nat Genet 25:232–234

Fang Y, Hearn S, Spector DL (2004) Tissue-specific expressionand dynamic organization of SR splicing factors in Arabid-opsis. Mol Biol Cell 15:2664–2673

Plant Mol Biol (2006) 62:637–653 651

123

Fortes P, Bilbao-Cortes D, Fornerod M, Rigaut G, Raymond W,Seraphin B, Mattaj IW (1999) Luc7p, a novel yeast U1snRNP protein with role in 5¢ splice site recognition. GeneDev 13:2425–2438

Fromont-Racine M, Rain JC, Legrain P (1997) Toward a func-tional analysis of the yeast genome through exhaustive two-hybrid screens. Nat Genet 16:277–282

Fu XD, Maniatis T (1992) Isolation of a complementary-DNAthat encodes the mammalian splicing factor SC35. Science256:535–538

Gatfield D, Unterholzner L, Ciccarelli FD, Bork P, Izaurralde E(2003) Nonsense-mediated mRNA decay in Drosophila: atthe intersection of the yeast and mammalian pathways.EMBO J 22:3960–3970

Gehring NH, Neu-Yilik G, Schell T, Hentze MW, Kulozik AE(2003) Y14 and hUpf3b form an NMD-activating complex.Mol Cell 11:939–949

Golovkin M, Reddy ASN (1996) Structure and expression of aplant U1 snRNP 70K gene: alternative splicing of U1snRNP 70K pre-mRNAs produces two different transcripts.Plant Cell 8:1421–1435

Golovkin M, Reddy ASN (1998) The plant U1 small nuclearribonucleoprotein particle 70K protein interacts with twonovel serine/arginine-rich proteins. Plant Cell 10:1637–1647

Golovkin M, Reddy ASN (1999) An SC35-like protein and anovel serine/arginine-rich protein interact with ArabidopsisU1-70K protein. J Biol Chem 274:36428–36438

Golovkin M, Reddy ASN (2003) Expression of U1 small nuclearribonucleoprotein 70K antisense transcript using APET-ALA3 promoter suppresses the development of sepals andpetals. Plant Physiol 132:1884–1891

Gozani O, Potashkin J, Reed R (1998) A potential role forU2AF-SAP 155 interactions in recruiting U2 snRNP to thebranch site. Mol Cell Biol 18:4752–4760

Groth C, Lardelli M (2002) The structure and function of ver-tebrate fibroblast growth factor receptor 1. Int J Dev Biol46:393–400

Halterman DA, Wei F, Wise RP (2003) Powdery mildew-in-duced Mla mRNAs are alternatively spliced and containmultiple upstream open reading frames. Plant Physiol131:558–567

Hanamura A, Caceres JF, Mayeda A, Franza BR, Krainer AR(1998) Regulated tissue-specific expression of antagonisticpre-mRNA splicing factors. RNA 4:430–444

Iggo RD, Jamieson DJ, Macneill SA, Southgate J, McPheat J,Lane DP (1991) P68 RNA helicase – identification of anucleolar form and cloning of related genes containing aconserved intron in yeasts. Mol Cell Biol 11:1326–1333

Kadowaki T, Hitomi M, Chen S, Tartakoff AM (1994) NuclearmRNA accumulation causes nucleolar fragmentation inyeast mtr2 mutant. Mol Biol Cell 5:1253–1263

Kalyna M, Lopato S, Barta A (2003) Ectopic expression ofatRSZ33 reveals its function in splicing and causespleiotropic changes in development. Mol Biol Cell14:3565–3577

Kataoka N, Bachorik JL, Dreyfuss G (1999) Transportin-SR, anuclear import receptor for SR proteins. J Cell Biol145:1145–1152

Kataoka N, Yong J, Kim VN, Velazquez F, Perkinson RA, WangF, Dreyfuss G (2000) Pre-mRNA splicing imprints mRNAin the nucleus with a novel RNA-binding protein that per-sists in the cytoplasm. Mol Cell 6:673–682

Kawaguchi R, Bailey-Serres J (2002) Regulation of translationalinitiation in plants. Curr Opin Plant Biol 5:460–465

Kohtz JD, Jamison SF, Will CL, Zuo P, Luhrmann R, Garcia-blanco MA, Manley JL (1994) Protein–protein interactions

and 5¢-splice-site recognition in mammalian messenger-RNA precursors. Nature 368:119–124

Kriventseva EV, Koch I, Apweiler R, Vingron M, Bork P,Gelfand MS, Sunyaev S (2003) Increase of functionaldiversity by alternative splicing. Trends Genet 19:124–128

Kuroyanagi N, Onogi H, Wakabayashi T, Hagiwara M (1998)Novel SR-protein-specific kinase, SRPK2, disassembles nu-clear speckles. Biochem Bioph Res Co 242:357–364

Lai MC, Lin RI, Huang SY, Tsai CW, Tarn WY (2000) A humanimportin-beta family protein, transportin-SR2, interactswith the phosphorylated RS domain of SR proteins. J BiolChem 275:7950–7957

Lalli E, Ohe K, Latorre E, Bianchi ME, Sassone-Corsi P (2003)Sexy splicing: regulatory interplays governing sex determi-nation from Drosophila to mammals. J Cell Sci 116:441–445

Lazar G, Schaal T, Maniatis T, Goodman HM (1995) Identifi-cation of a plant serine-arginine-rich protein similar to themammalian splicing factor SF2/ASF. Proc Natl Acad SciUSA 92:7672–7676

Le Hir H, Gatfield D, Izaurralde E, Moore MJ (2001) The exon-exon junction complex provides a binding platform forfactors involved in mRNA export and nonsense-mediatedmRNA decay. EMBO J 20: 4987–4997

Lei EP, Silver PA (2002) Protein and RNA export from thenucleus. Dev Cell 2:261–272

Li S, Armstrong CM, Bertin N, Ge H, Milstein S, Boxem M,Vidalain PO, Han JD, Chesneau A, Hao T, Goldberg DS, LiN, Martinez M, Rual JF, Lamesch P, Xu L, Tewari M, WongSL, Zhang LV, Berriz GF, Jacotot L, Vaglio P, Reboul J,Hirozane-Kishikawa T, Li Q, Gabel HW, Elewa A, Baum-gartner B, Rose DJ, Yu H, Bosak S, Sequerra R, Fraser A,Mango SE, Saxton WM, Strome S, Van Den Heuvel S,Piano F, Vandenhaute J, Sardet C, Gerstein M, Doucette-Stamm L, Gunsalus KC, Harper JW, Cusick ME, Roth FP,Hill DE, Vidal M (2004) A map of the interactome networkof the metazoan C. elegans. Science 303:540–543

Locatelli F, Bracale M, Magaraggia F, Faoro F, Manzocchi LA,Coraggio I (2000) The product of the rice myb7 unsplicedmRNA dimerizes with the maize leucine zipper Opaque2and stimulates its activity in a transient expression assay.J Biol Chem 275:17619–17625

Lopato S, Gattoni R, Fabini G, Stevenin J, Barta A (1999a) Anovel family of plant splicing factors with a Zn knucklemotif: examination of RNA binding and splicing activities.Plant Mol Biol 39:761–773

Lopato S, Kalyna M, Dorner S, Kobayashi R, Krainer AR, BartaA (1999b) atSRp30, one of two SF2/ASF-like proteins fromArabidopsis thaliana, regulates splicing of specific plantgenes. Gene Dev 13:987–1001

Lopato S, Forstner C, Kalyna M, Hilscher J, Langhammer U,Indrapichate K, Lorkovic ZJ, Barta A (2002) Network ofinteractions of a novel plant-specific Arg/Ser-rich protein,atRSZ33, with atSC35-like splicing factors. J Biol Chem277:39989–39998

Lopez AJ (1998) Alternative splicing of pre-mRNA: develop-mental consequences and mechanisms of regulation. AnnuRev Genet 32:279–305

Lorkovic ZJ, Barta A (2002) Genome analysis: RNA recognitionmotif (RRM) and K homology (KH) domain RNA-bindingproteins from the flowering plant Arabidopsis thaliana.Nucleic Acids Res 30:623–635

Lorkovic ZJ, Lopato S, Pexa M, Lehner R, Barta A (2004)Interactions of Arabidopsis RS domain containing cyclo-philins with SR proteins and U1 and U11 snRNP-specificproteins suggest their involvement in pre-mRNA splicing. JBiol Chem 279:33890–33898

652 Plant Mol Biol (2006) 62:637–653

123

Lynch KW, Maniatis T (1996) Assembly of specific SR proteincomplexes on distinct regulatory elements of the Drosophiladoublesex splicing enhancer. Gene Dev 10:2089–2101

Magaraggia F, Solinas G, Valle G, Giovinazzo G, Coraggio I(1997) Maturation and translation mechanisms involved inthe expression of a myb gene of rice. Plant Mol Biol35:1003–1008

Manley JL, Tacke R (1996) SR proteins and splicing control.Gene Dev 10:1569–1579

Mohr SE, Dillon ST, Boswell RE (2001) The RNA-bindingprotein Tsunagi interacts with Mago Nashi to establishpolarity and localize oskar mRNA during Drosophilaoogenesis. Gene Dev 15:2886–2899

Montag K, Salamini F, Thompson RD (1995) ZEMa, a memberof a novel group of MADS box genes, is alternatively splicedin maize endosperm. Nucleic Acids Res 23:2168–2177

Mortillaro MJ, Berezney R (1998) Matrin CYP, an SR-rich cy-clophilin that associates with the nuclear matrix and splicingfactors. J Biol Chem 273:8183–8192

Mount SM, Salz HK (2000) Pre-messenger RNA processingfactors in the Drosophila genome. J Cell Biol 150:F37-F43

Murray MV, Kobayashi R, Krainer AR (1999) The type 2C Ser/Thr phosphatase PP2C gamma is a pre-mRNA splicingfactor. Gene Dev 13:87–97

Nash J, Walbot V (1992) Bronze-2 gene-expression and intronsplicing patterns in cells and tissues of Zea-Mays L. PlantPhysiol 100:464–471

Okanami M, Meshi T, Iwabuchi M (1998) Characterization of aDEAD box ATPase/RNA helicase protein of Arabidopsisthaliana. Nucleic Acids Res 26:2638–2643

Quesada V, Macknight R, Dean C, Simpson GG (2003) Auto-regulation of FCA pre-mRNA processing controls Arabid-opsis flowering time. EMBO J 22:3142–3152

Reddy ASN (2001) Nuclear pre-mRNA splicing in plants. CritRev Plant Sci 20:523–571

Reddy ASN (2004) Plant serine/arginine-rich proteins and theirrole in pre-mRNA splicing. Trends Plant Sci 9:541–547

Rydberg B, Spur B, Karran B (1990) cDNA cloning and chro-mosomal assignment of the human O6-methylguanine-DNAmethyltransferase. cDNA expression in Escherichia coli andgene expression in human cells. J Biol Chem 265:9563–9569

Ryner LC, Baker BS (1991) Regulation of Doublesex pre-mes-senger-RNA processing occurs by 3¢-splice site activation.Gene Dev 5:2071–2085

Savaldi-Goldstein S, Aviv D, Davydov O, Fluhr R (2003)Alternative splicing modulation by a LAMMER kinaseimpinges on developmental and transcriptome expression.Plant Cell 15:926–938

Screaton GR, Caceres JF, Mayeda A, Bel MV, Plebanski M,Jackson DG, Bell JI, Krainer AR (1995) Identification andcharacterization of 3 members of the human SR family ofpre-messenger-RNA splicing factors. EMBO J 14:4336–4349

Senger B, Simos G, Bischoff FR, Podtelejnikov A, Mann M,Hurt E (1998) Mtr10p functions as a nuclear import receptorfor the mRNA-binding protein Np13p. EMBO J 17:2196–2207

Siebel CW, Feng LN, Guthrie C, Fu XD (1999) Conservation inbudding yeast of a kinase specific for SR splicing factors.Proc Natl Acad Sci USA 96:5440–5445

Singh G, Lykke-Andersen J (2003) New insights into the for-mation of active nonsense-mediated decay complexes.Trends Biochem Sci 28:464–466

Stanyon CA, Finley RL Jr (2000) Progress and potential ofDrosophila protein interaction maps. Pharmacogenomics1:417–431

Sun YJ, Flannigan BA, Madison JT, Setter TL (1997) Alterna-tive splicing of cyclin transcripts in maize endosperm. Gene195:167–175

Tacke R, Tohyama M, Ogawa S, Manley JL (1998) Human Tra2proteins are sequence-specific activators of pre-mRNAsplicing. Cell 93:139–148

Tuteja N (2000) Plant cell and viral helicases: essential enzymesfor nucleic acid transactions. Crit Rev Plant Sci 19:449–478

Wang CY, Chua K, Seghezzi W, Lees E, Gozani O, Reed R(1998a) Phosphorylation of spliceosomal protein SAP 155coupled with splicing catalysis. Gene Dev 12:1409–1414

Wang HY, Lin W, Dyck JA, Yeakley JM, Zhou SY, Cantley LC,Fu XD (1998b) SRPK2: a differentially expressed SR pro-tein-specific kinase involved in mediating the interactionand localization of pre-mRNA splicing factors in mamma-lian cells. J Cell Biol 140:737–750

Weber H, Borisjuk L, Heim U, Buchner P, Wobus U (1995) Seedcoat-associated invertases of fava bean control bothunloading and storage functions: cloning of cDNAs and celltype-specific expression. Plant Cell 7:1835–1846

Wu JY, Maniatis T (1993) Specific interactions between proteinsimplicated in splice-site selection and regulated alternativesplicing. Cell 75:1061–1070

Xie J, Beickman K, Otte E, Rymond BC (1998) Progressionthrough the spliceosome cycle requires Prp38p function forU4/U6 snRNA dissociation. EMBO J 17:2938–2946

Zamore PD, Patton JG, Green MR (1992) Cloning and domain-structure of the mammalian splicing factor U2AF. Nature355:609–614

Zhang WQ, Yuan YF, Wang YN, Yuan Z, Mi HF, He BL (2002)Human cyclophilin 33 (hCyP33) in T-cell binds specificallyto poly(A)(+)RNA (mRNA). Sci China Ser B 45:275–281

Zhou Z, Licklider LJ, Gygi SP, Reed R, Sim J, Griffith J (2002)Comprehensive proteomic analysis of the human spliceo-some: purification and electron microscopic visualization offunctional human spliceosomes. Nature 419:182–185

Zolotukhin AS, Tan W, Bear J, Smulevitch S, Felber BK (2002)U2AF participates in the binding of TAP (NXF1) tomRNA. J Biol Chem 277:3935–3942

Plant Mol Biol (2006) 62:637–653 653

123