Sense and antisense Foxl2 transcripts in mouse

www.elsevier.com/locate/ygeno

Genomics 85 (20

Sense and antisense Foxl2 transcripts in mouse

Julie Cocqueta, Maelle Pannetierb, Marc Fellousa, Reiner A. Veitiaa,TaINSERM E0021 and U361, Hopital Cochin, Pavillon Baudelocque, 123 Boulevard de Port Royal, 75014 Paris, France

bBiologie du Developpement et Reproduction, INRA Jouy-en-Josas, France

Received 13 December 2004; accepted 20 January 2005

Abstract

FOXL2 is a forkhead transcription factor involved in eyelid development and in the development and adult function of the ovary in

mammals. In mouse, we have previously suggested the existence of two mRNA isoforms of Foxl2 that result from an alternative

polyadenylation. In this study, we characterize in depth the structure and expression of these two variants. We also describe an antisense

transcript that overlaps the whole Foxl2 transcription unit. This antisense transcript, called Foxl2OS (for opposite strand), yields several

isoforms resulting from alternative splicing. No significant coding region was found in the Foxl2OS sequence. Foxl2OS displays a pattern of

expression very similar to that of Foxl2 in the gonads during development and at the adult age. RNA FISH experiments show that both

transcripts are expressed in the same cells at the same time. We suggest that Foxl2OS is a noncoding antisense RNA that may be involved in

the regulation of Foxl2. All in all our results provide new insights about the organization of the murine Foxl2 locus. This might help us

understand its regulation and function.

D 2005 Elsevier Inc. All rights reserved.

Keywords: FOXL2; Antisense RNA; Ovarian development; Alternative splicing; Alternative polyadenylation

FOXL2 is a winged helix/forkhead domain transcription

factor involved in the early development and function of the

adult mammalian ovary as well as in eyelid development [1].

Its mutations in human are responsible for a rare genetic

disease characterized by eyelid malformation, associated or

not with premature ovarian failure (blepharophimosis syn-

drome of types I and II, respectively; OMIM 110100) [2,3].

The nucleotide and amino acid sequences of FOXL2 have

been shown to be very conserved in numerous vertebrates,

ranging from fish to human [4]. Its gonadal expression is also

conserved among vertebrates: The FOXL2 protein is

expressed throughout ovarian development and is one of

the earliest known sex dimorphic marker of ovarian

determination/differentiation (i.e., no substantial expression

in the male gonads). Its ovarian expression (studied in detail

in mammals) seems to be strictly somatic, begins early in

development, and persists until the adult age [4]. However, in

mouse, Loffler and colleagues have detected Foxl2 tran-

0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygeno.2005.01.007

T Corresponding author. Fax: +33 1 43 26 44 08.

E-mail address: [email protected] (R.A. Veitia).

scripts in several oocytes [5]. An ovarian expression profile

similar to that observed in mammals has been detected in the

red-eared slider turtle [5], the chicken [6], and the rainbow

trout [7]. Thus, FOXL2 is though to be a key factor of early

ovarian development and of the adult ovarian function in

vertebrates. Foxl2 has been knocked out in mouse [8,9]. As

expected, homozygous Foxl2�/� female mice are infertile.

Interestingly, the defect lies in the granulosa cell differ-

entiation, which is arrested at an early stage of folliculo-

genesis. This provokes massive apoptosis resulting in

depletion of the initial pool of follicles and in ovarian atresia.

These results suggest that Foxl2 plays an important role in

granulosa cell differentiation and ovary maintenance [8,9].

In the present study, we characterized two murine Foxl2

transcripts resulting from alternative polyadenylation [4].

The structural and expressional characteristics of these two

transcripts (named Foxl2-short and Foxl2-long) were

studied in detail. In addition, we identified an antisense

RNA that overlaps the whole Foxl2 transcription unit. This

antisense RNA, called Foxl2OS (for opposite strand), was

found to encompass several isoforms as a result of

05) 531–541

J. Cocquet et al. / Genomics 85 (2005) 531–541532

alternative splicing. We characterized Foxl2OS transcripts

and studied thoroughly their pattern of expression. We also

studied Foxl2 and Foxl2OS expression by RNA fluores-

cence in situ hybridization (FISH). We hypothesize that

Foxl2OS is involved in the regulation of Foxl2.

Results and discussion

Murine Foxl2 is expressed as two isoforms resulting from

differential polyadenylation

Our in silico analyses have led us to detect several

expressed sequence tags (ESTs) from mouse ovary or eyelid/

eyeball indicating the existence of two polyadenylation sites

in murine Foxl2 [4]. Several ESTs end at a canonic poly-

adenylation signal (AAUAAA). This site is orthologous to

the site found in human [2] and rat transcripts. However, other

mouse ESTs were found to end 430 nucleotides downstream

of this site, involving a noncanonical polyadenylation signal

(UAUAAA). In the rat, two ESTs end around the non-

canonical signal (AAGAAU) located 400 nucleotides down-

stream of the first polyadenylation site [4]. In human, there is

no EST evidence indicating the existence of such a longer

Fig. 1. (A) Expression of Foxl2-short and Foxl2-long mRNAs. RT-PCR experime

parallel, we performed RT-PCR on h2-microglobulin (B2M) to normalize the resu

Foxl2-long to detect striking (presence/absence) differences between these tissues.

of reverse transcriptase. I, intestine; M, skeletal muscle; K, kidney; Li, liver; Lu, lun

in the ovary and the eyelid. (B) Northern blot of Foxl2 polyadenylation isoforms.

beginning of Foxl2 ORF. O, ovary total RNA (10 Ag). A, AT29C cells poly(A)+ RN

They correspond to Foxl2-short and Foxl2-long transcripts, respectively. (C) Fluo

Foxl2sensPE1 reverse primer was used to extend Foxl2 RNA from AT29C cells. 1 Aprior to being loaded onto the sequencing gel. The lengths of the extension product

points to a peak of the marker (i.e., 94, 109, 116, 172, or 186 nt). The two arrows po

ATG start codon). The extension products are 124 and 139 nt long, respectively.

transcript. This is in agreement with previous Northern blot

experiments in which a single FOXL2 band was obtained [2].

The mouse ESTs mentioned above suggest that both

polyadenylation isoforms are apparently expressed in the

same tissues (i.e., ovary, eyelid/eyeball, pituitary gland).

These tissues are known to express the Foxl2 protein [4].

Reverse transcriptase-polymerase chain reaction (RT-PCR)

on several mouse tissues confirmed our in silico observa-

tion: both isoforms are expressed in the ovary and the

eyelid/eyeball (Fig. 1A). As expected, no signal was found

in other tissues, where no RNA or protein expression has

ever been found, such as muscle, intestine, kidney, liver, and

lung (Fig. 1A). We then performed Northern blot experi-

ments on RNA extracted from mouse ovary and a mouse

granulosa cell line, AT29C [10], with a probe designed to

recognize the Foxl2 open reading frame (ORF). In both

samples, two bands of about 2.8 and 3.3 kb were detected

(Fig. 1B). Northern blot results suggest that both isoforms

are expressed at similar levels, in the ovary and in AT29C

cells. The sizes of the bands (i.e., 2.8 and 3.3 kb) are

compatible with the expected lengths of one transcript

orthologous to the reported human transcription unit (Foxl2-

short) and another polyadenylated transcript containing 430

additional nucleotides (Foxl2-long). Therefore, both iso-

nts were performed on RNA extracted from several tissues of adult mice. In

lts. Thirty-three cycles of PCR were performed to amplify Foxl2-short and

Thirty cycles were performed for B2M. +/� symbolize the presence/absence

g; E, eyelid; O, ovary. Foxl2-short and Foxl2-long mRNAs are detected only

The membrane was hybridized with a 381-bp probe that corresponds to the

A (8 Ag). In both samples, two bands of about 2.8 and 3.3 kb were detected.

rescent primer extension to detect Foxl2 transcriptional start site(s). 5VFAM-

l of Genscan-2500 ROX (ABI fluorescent marker) was added to each sample

s were calculated with respect to the peaks of ROX marker. Each arrowhead

int to Foxl2 transcriptional start sites�228 and�243 (with respect to Foxl2

A control reaction, without reverse transcriptase, is shown in light gray.

J. Cocquet et al. / Genomics 85 (2005) 531–541 533

forms contain the ORF and could be translated to produce

Foxl2 protein. 3V rapid amplification of cDNA ends

(3VRACE) on RNA extracted from mouse ovary and

AT29C cells confirmed the existence of these two isoforms

and of their polyadenylation (data not shown, available

upon request). We called these two Foxl2 transcripts Foxl2-

short and Foxl2-long.

We performed 5VRACE experiments and primer exten-

sion to identify the transcriptional start sites (TSS) of mouse

Foxl2. By 5VRACE, four specific bands of approximately

100, 150, 190, and 210 bp were obtained. They correspond

to four putative TSS located at �136, �189, �228, and

�243 with respect to the Foxl2 translation start codon

(ATG) (data not shown). When we performed primer

extension on AT29C RNA with a primer carrying a

fluorescent label, the lengths of the resulting extension

products (i.e., 85, 124, and 139 nucleotides) corresponded to

three of the four TSS found by 5VRACE (TSS �189, �228,

and �243) (Fig. 1C). The structures of Foxl2 transcripts

deduced from this analysis are shown in Fig. 2A.

Both Foxl2 polyadenylation isoforms present similar

expression patterns in the developing gonad

Messenger RNAs are sometimes differentially polyade-

nylated during the cell cycle or in a tissue-specific or

developmentally specific pattern [11]. Our results indicate

that both Foxl2 transcripts (short and long) are presumed to

Fig. 2. (A) Organization of Foxl2 transcripts. The structures of Foxl2-short and Fo

indicate Foxl2 polyadenylation sites 1 and 2 (pA1 and pA2, respectively) and tran

are represented by dotted lines. (B) Organization of Foxl2OS transcripts. The p

transcripts, deduced from our results, are displayed. Vertical arrows indicate the Fo

indicate the alternative exons of Foxl2OS that were detected. The Foxl2OS segmen

that we have used to amplify Foxl2OS) to +2488 (the 5V-most Foxl2OS TSS w

(represented by double oblique bars) of Foxl2OS transcripts remains to be resolv

Foxl2OS-2R (2R), Foxl2OS-3R (3R), Foxl2-PE1, Foxl2-ORF-1R, Foxl2OS-PE1

region of Foxl2OS.

contain the Foxl2 ORF and to have similar expression

patterns. We studied the expression profiles of both Foxl2

mRNA isoforms during development and after birth, in the

gonad. Specifically, we performed fluorescence semiquanti-

tative RT-PCR on RNA extracted from mouse male and

female gonads at several developmental stages (11.5, 12.5,

and 16 days postcoitum (dpc) and after birth, 5 and 40 days

postpartum (dpp)). In female gonads, we detected Foxl2-

short and Foxl2-long expression very faintly from 12.5 dpc

and then more strongly at 16 dpc. The PCR signal increased

throughout development and persisted until the adult age. No

substantial expression was observed in male gonads at any

stage (Fig. 3). These results are in line with the pattern of

expression of the Foxl2 protein [1] and indicate a coex-

pression of both transcripts and the protein. In summary, both

mRNA isoforms are apparently expressed at the same time

and in the same tissues, and both can encode the entire Foxl2

protein. In rat, as suggested by two ESTs, there is also a

further polyadenylation site, located 400 nucleotides down-

stream of the first one. In human, there is no evidence for the

use of such an alternative site. The role, if any, of alternative

polyadenylation in the context of Foxl2 remains to be

determined. This phenomenon could be specific to rodents.

Identification of an antisense transcript to Foxl2: Foxl2OS

We have found in silico evidence for the existence of a

sequence transcribed from the opposite strand of Foxl2.

xl2-long transcripts, deduced from our results, are displayed. Vertical arrows

scriptional start sites (TSS). The Foxl2 start (AUG) and stop (UGA) codons

utative structures of the longest known and of the most spliced Foxl2OS

xl2OS TSS described in the text. Dotted rectangles (numbered from 1 to 4)

t we have studied spans from �1929 (i.e., the location of the most 3Vprimer

e have observed). Note that the detailed organization of the inner region

ed. Horizontal arrows show the locations of the primers, Foxl2OS-2F (2F),

, and FOXL2-B [2], that have been used to determine the structure of this

Fig. 3. Temporal expression of Foxl2-short and Foxl2-long in gonads

throughout development. Fluorescence RT-PCR was performed on RNAs

from male and female gonads of 11.5, 12.5, and 16 dpc and 5 and 40 dpp.

Foxl2-short and Foxl2-long were amplified by 23 cycles of PCR to

preserve the semiquantitative character of our approach. At early stages, a

contamination of the gonad with mesonephros cannot be excluded.

Fluorescent RT-PCR products were quantified and normalized with respect

to B2M amplification product performed in the same tube. Foxl2 short and

long isoforms are very faintly detected from 12.5 dpc in female gonads.

Then, their expression increases throughout development until the adult

age. No significant expression could be observed in male gonads of any

stage.

Table 1

Exon/intron junctions in Foxl2OS

Donor and acceptor sites of the four splice events of Foxl2OS that were

found in the upstream region of Foxl2. The DNA sequences are directed

from left to right in the 5V-to-3Vorientation of Foxl2OS. Each number above

the sequences indicates the location of the junction with respect to the first

ATG codon of Foxl2 (cf. Fig 2B). Exons are represented in uppercase and

introns (or alternative exons) in lowercase. The numbering of the introns or

exons refers to those that were characterized in depth.


Specifically, several ESTs (GenBank Accession Nos.

BB617689, BB666395, BM461091), when contiged, were

found to correspond to a region that spans from �1.8 kb (+1

being the first ATG codon of Foxl2) to the transcriptional

unit of Foxl2. One of these ESTs (GenBank Accession No.

BB617689) results from splicing using canonical sites (i.e.,

GT/AG) that can work only in the opposite orientation with

respect to the Foxl2 transcriptional unit known so far (cf.

Table 1). This EST sequence covers the region from �1789

to �863 bp and lacks two spliced fragments (from �1542 to

�1458 bp and from �1426 to �1257 bp). Some of these

ESTs come from ovarian tissue. To confirm the existence of

an antisense transcript(s), we performed RT-PCR experi-

ments on RNA extracted from adult mouse ovary. Reverse

transcription was carried out using an oligonucleotide

(FoxlOS-3R, Fig. 2B) designed to prime the reaction only

from transcripts that are antisense with respect to Foxl2.

PCR was then performed using a pair of primers (Foxl2-

ORF-1R and Foxl2OS-2R, Fig. 2B) that flanks a large

region where the splice events were expected. Seven bands

were obtained and sequenced (Fig. 4A). They result from

the alternative splicing of four segments. This confirms the

existence of two alternative exons as suggested by the ESTs

and shows the existence of two other alternative exons. We

called this set of transcripts Foxl2OS, as they are transcribed

on the strand opposite to Foxl2. The sequence and the

location of the identified splice sites are shown on Table 1

and Fig. 2B.

Characterization of Foxl2OS transcripts

We performed 5V and 3VRACE and primer extension

experiments to characterize Foxl2OS transcripts ends. With

5VRACE, we found two potential TSS. Interestingly, they

are both located very close to the first Foxl2 polyadeny-

lation site, at 33 and 55 nucleotides downstream of the

polyadenylation site, respectively (cf. Fig. 2B). Fluorescent

primer extension with one primer located in this region

showed two extension products of 93 and 115 nt that

correspond to the two TSS identified by 5VRACE (Fig. 4B).

Our results suggest that the 5V end of some (if not all)

Foxl2OS isoforms is located around the end of the Foxl2-

short sense transcription unit. However, Foxl2OS tran-

scription might start beyond the two potential TSS that

have been described here. One cannot exclude that a

secondary structure of the RNA is formed in this region

and stops the progression of the reverse transcriptase

(affecting primer extension and 5VRACE experiments).

Another possibility is the formation of a double-stranded

RNA duplex that results from the annealing of Foxl2-short

and Foxl2OS transcripts. In this case, the Foxl2OS 5Vprotruding end might be degraded, leading to an apparent

start of Foxl2OS transcripts where Foxl2-short transcripts

end. The identification of potential TSS of Foxl2OS where

Foxl2-short transcript ends argues in favor of a role for

Foxl2OS in the stabilization/destabilization of Foxl2

mRNA (via its 3VUTR) or in the regulation of the

polyadenylation of Foxl2-short.

Fig. 4. (A) Foxl2OS RT-PCR. RT-PCR were performed on adult mouse ovary total RNA. O+, ovary cDNA. O�, control without reverse transcriptase. We used

a pair of primers that flanks the four alternative exons of Foxl2OS (i.e., Foxl2-ORF-1R and Foxl2OS-2R). The seven observed bands correspond to

alternatively spliced variants of Foxl2OS. (B) Foxl2OS Fluorescent primer extension. Foxl2OS-PE1 reverse primer was used to extend Foxl2OS transcripts

from AT29C cells, as described for Foxl2 (cf. Fig. 1C). Each arrowhead points to a peak of the marker (i.e., 94, 109, 116, 172, or 186 nt). The two arrows point

to potential Foxl2OS TSS at approx +33 and +55 bp with respect to the Foxl2 first polyadenylation site. The extension products are 93 and 115 nt long,

respectively. (C) Northern blot of Foxl2OS. The membrane was hybridized with a 381-nt asymmetric (essentially single stranded) probe that corresponds to the

beginning of the Foxl2 ORF and designed to detect only Foxl2OS RNAs. O, ovary total RNA (10 Ag); A, AT29C cells poly(A)+ RNA (8 Ag). In the total RNAsample, a 4.4-kb band can be detected, which is absent in the poly(A)+ RNA sample. (D) Expression of Foxl2OS transcripts. RT-PCR experiments were

performed on RNA extracted from several tissues of adult mice. We used primers that flank Foxl2OS alternative exons 3 and 4 (primers Foxl2OS-2F and 2R) to

detect a possible difference of representation of the isoforms between the tissues analyzed. In parallel, we performed RT-PCR on B2M to normalize the results.

Thirty-five cycles of PCR were done to amplify Foxl2OS to detect striking (presence/absence) differences between these tissues. Thirty cycles were performed

for B2M. +/� symbolize the presence/absence of reverse transcriptase. I, intestine; M, skeletal muscle; K, kidney; Li, liver; Lu, lung; E, eyelid; O, ovary.

Foxl2OS isoforms are detected only in the ovary and the eyelid.


Our attempts to determine the Foxl2OS 3Vend by RACE

experiments were unsuccessful. Using primers scattered

from the Foxl2 ORF up to �2.5 kb (with respect to the first

ATG codon of Foxl2), no specific RACE products could be

obtained. Two reasons may explain this: (1) the 3VRACEexperiment was performed to detect 3Vends of polyadeny-lated RNA, as the primer used for reverse transcription was

an oligo(dT). Like many other antisense RNAs (i.e., human

N-myc antisense, murine mbp gene antisense) [12], Fox-

l2OS might not be polyadenylated. (2) Foxl2OS RNA could

be polyadenylated but might be longer than expected. Some

ESTs (for example, GenBank Accession No. BB021606) are

located approximately �6.5 kb upstream of the ATG start

codon of Foxl2 (i.e., about 9 kb away from Foxl2OS

putative TSS). They end at a canonical polyadenylation site

with the same orientation as Foxl2OS. In addition, two other

ESTs (GenBank Accession Nos. BB465328 and BB600376)

were found to cover the regions from �3 to �2.3 kb and

from �3.7 to �3.1 kb, respectively. These ESTs could either

correspond to another transcript in this region or be the 3Vend of Foxl2OS RNA. To test the latter hypothesis, which

implies that Foxl2OS is about 9 kb long, we performed

3VRACE experiments with primers closer to the polyadeny-

lation site identified in the EST database. Indeed, we

confirmed the existence of a transcript that ends at this

polyadenylation site in the ovary. However, as the distance

between this site and the Foxl2OS last known exon is 4 kb,

we could not detect a PCR product joining Foxl2OS and this

downstream polyadenylation site. Consequently, we have no

evidence indicating that Foxl2OS and this polyadenylated

transcript are the same RNA entity.

In addition, we performed Northern blots on total mouse

ovary RNA and AT29C poly(A)+ RNA using an asym-

metric probe designed to detect only Foxl2OS. In the total

RNA sample, we observed a 4.4-kb broad band that could

not be detected in the poly(A)+ RNA sample (Fig. 4C). This

result tends to favor the hypothesis that Foxl2OS is not

polyadenylated. In addition, a length of 4.4 kb suggests that

Foxl2OS ends about 1 kb downstream of the last alternative

exon detected, to be consistent with the putative location of

its TSS. We have detected seven Foxl2OS isoforms with a

maximum difference in length of 860 bp (at least in the


well-characterized region where splicing events take place).

This could explain the width of the band in the Northern

blot. However, we cannot exclude that one of these isoforms

is 9 kb long but too poorly expressed to be detectable. The

observed 4.4 kb band was faint (exposure of the X-ray film

for several weeks). This might explain why in our previous

Northern blot with a double-stranded Foxl2 probe we did

not detect any Foxl2OS signal. Foxl2OS may be less

expressed than Foxl2 transcripts and/or its signal might be

diluted because of the number of its splice variants. No

obvious coding region was found in Foxl2OS (Genscan

prediction program), even when the analysis was extended

to the larger 9-kb region mentioned above.

Foxl2 and Foxl2OS transcripts possess similar patterns of

expression

Our preliminary data indicated that Foxl2OS is expressed

at least in adult ovary. Here, we performed RT-PCR on

several mouse tissues. We used primers that flank the

alternative exons 3 and 4 of Foxl2OS (Foxl2OS-2F and 2R,

cf. Fig. 2B), to detect a possible difference of representation

of these alternative transcripts. Four bands (that we named

A, B, C, and D) corresponding to alternatively spliced

Foxl2OS isoforms were amplified. Amplicon A is 319 bp in

length and corresponds to transcripts having alternative

exons 3 and 4 spliced; B (404 bp) corresponds to transcripts

having alternative exon 3 spliced, C (487 bp) to transcripts

having alternative exon 4 spliced, and D (572 bp) to

transcripts with alternative exons 3 and 4 retained. We

observed the expression of all Foxl2OS isoforms in the

eyelid and the ovary. No Foxl2OS could be detected in other

tested tissues (i.e., muscle, intestine, kidney, liver, and lung)

(Fig. 4D). Similar results have been obtained with primers

that flank alternative exons 1 and 2 of Foxl2OS (data not

shown). Thus, Foxl2OS was detected in the same tissues

that express Foxl2 transcripts (short and long isoforms) and

protein [4].

Fig. 5. Temporal expression of Foxl2OS in gonads throughout development. Fluor

Fig. 3. We used primers that flank Foxl2OS alternative exons 3 and 4 (primers Fox

of Foxl2OS amplicon A (319 bp), B (404 bp), C (487 bp), and D (572 bp) is show

from 12.5 dpc, which increases at 16 dpc and is maintained until the adult age. In

We next studied the temporal expression of Foxl2OS in

mouse male and female gonads by fluorescent semi-

quantitative PCR experiments. We used the first pair of

primers described above (i.e., Foxl2OS-2F and 2R), to

explore if the expression of Foxl2OS variants evolved

differently throughout development and after birth. All the

observed Foxl2OS transcripts displayed similar patterns of

expression. In female gonads, Foxl2OS transcripts were

expressed at a low level from 12.5 dpc, increased at 16 dpc,

and were maintained until the adult age. In male gonads, as

observed for Foxl2 transcripts, no substantial expression

could be found at any stage of development or after birth

(Fig. 5). Thus, Foxl2OS isoforms show a pattern of

expression that is very similar to that of Foxl2 transcripts

and protein.

RNA FISH experiments in a granulosa cell line reveal that

the same cells express Foxl2 and Foxl2OS transcripts

To study further Foxl2 and Foxl2OS expression, we

performed RNA FISH experiments on the AT29C cell line.

The purpose of these experiments was to determine whether

Foxl2 and Foxl2OS are expressed within the same cells. To

perform RNA FISH we used single-stranded probes that

were differentially labeled (fluorescein isothiocyanate

(FITC), to detect Foxl2 mRNA, and Texas red to detect

Foxl2OS RNA) and directed against different regions of the

Foxl2 locus to avoid probe-to-probe annealing. The results

showed that Foxl2 and Foxl2OS transcripts were present in

the same cells (Fig. 6A). We could also observe differences

in Foxl2 and Foxl2OS level of expression from cell to cell.

This could be due to the presence of several clones in the

cell culture or to differential expression, for example,

through cell cycle.

To confirm the specificity of the hybridizations, we

performed experiments on L929 cells. This cell line is

derived from adult mouse lung and does not express either

Foxl2 or Foxl2OS (RT-PCR results, data not shown). FISH

escent RT-PCR was performed on male and female gonads, as described for

l2OS-2F and 2R) and performed 25 cycles of PCR. The normalized intensity

n. In female gonads, Foxl2OS transcripts are expressed at a very faint level

male gonads, no significant level of expression could be found at any stage.

Fig. 6. RNA fluorescence in situ hybridization. For all the RNA FISH experiments, the same experimental procedure and image acquisition conditions were

used. Cell nuclei were stained in blue with Hoechst. RNA FISH experiments were performed with differentially labeled probes. (A) RNA FISH on AT29C cells

to detect Foxl2 and Foxl2OS transcripts. Foxl2 transcripts in green (FITC) and Foxl2OS in red (Texas red) are detected within the same cells, mainly in the

cytoplasm (particularly in the perinuclear region). This expression is not homogeneous from cell to cell, as they do not express Foxl2 and Foxl2OS with the

same intensity. (B) RNA FISH performed on an irrelevant cell line (L929). Only faint signals (attributable to a background nonspecific hybridization) could be

observed for both probes designed to detect Foxl2(green) and Foxl2OS transcripts (red). (C) RNA FISH on AT29C cells with two types of irrelevant probes

(green and red). Only background signals could be observed. (D) RNA FISH performed on AT29C cells with a probe that detects G3PDH transcripts (green).

G3PDH mRNA expression level was comparable in all the AT29C cells.


performed on these cells gave faint signals for both probes,

attributable to a background nonspecific hybridization (Fig.

6B). Similar background signals were obtained in AT29C

cells with irrelevant probes (that correspond to antiparallel

sequences of Foxl2OS oligonucleotide probes and antipar-

allel sequence of B2M probe) (Fig. 6C).


To detect potential differences in treatment that would

explain differences in Foxl2 and Foxl2OS levels of

expression from cell to cell, we probed the glyceraldehyde-

3-phosphate dehydrogenase (G3PDH) mRNA. G3PDH

expression level was comparable in all the AT29C cells

(Fig. 6D). We conclude: (i) that the expression of Foxl2 and

Foxl2OS in AT29C cells is not homogeneous and (ii) that

Foxl2 and Foxl2OS are expressed in the same cells and both

are mostly detected in the cytoplasm, particularly in the

perinuclear region. Our previous results indicate that the

AT29C murine granulosa cell line is, in many respects

concerning the locus Foxl2, similar to the ovarian granulosa

cells. Thus, our RNA FISH results might be extrapolated to

the mouse granulosa.

As mentioned above, no potential coding region was

detected in the Foxl2OS sequence, thus its cytoplasmic

location might be intriguing at first sight for a presum-

ably nontranslatable transcript. Antisense-mediated regu-

lation is generally described as a cytoplasmic event,

operating mostly at the level of messenger stability by

the formation of RNA–RNA duplexes (for review, see

[13]). For example, N-myc antisense transcripts that are

nonpolyadenylated have been found in the cytoplasm

and, interestingly, most of them were found to exist in

RNA–RNA duplexes with approximately 5% of the sense

N-myc mRNA [14] (for review see [12]). Moreover,

although controversial, Kimelman and Kirschner [15]

have shown that Xenopus bFGF antisense transcript

participates in the editing of the bFGF mRNA sequence

during maturation of the oocyte. As this activity occurs

only on double-stranded RNA, they inferred the for-

mation of a sense–antisense RNA duplex in the oocyte.

Colocalization of Foxl2OS and Foxl2 transcripts might

result in the formation of cytoplasmic RNA–RNA

duplexes.

In conclusion, we have shown that Foxl2 and Foxl2OS

are expressed in the same tissues. In addition, they are

apparently coexpressed at the cellular level. Two possibi-

lities could explain this finding. (1) Coregulation may

concern many genes located in this region and thus may

not be restricted to Foxl2 and Foxl2OS. If so, Foxl2OS

function (if any) may be independent of that of Foxl2. The

tissue-specific expression of all the genes (except Foxl2OS)

in 1 Mb around Foxl2 has been previously studied in

mouse and human. The expression of only one gene (i.e.,

Faim, Fas apoptotic inhibitory molecule) was similar to

that of Foxl2 [16]. This result does not favor the hypothesis

of a general coregulation of the genes located in this

region. (2) Foxl2 and Foxl2OS coexpression may also be

explained by an involvement of Foxl2OS in the regulation

of Foxl2. This is further suggested by the absence of any

substantial coding region in Foxl2OS, its cytoplasmic

location, and the fact that it covers the whole transcription

unit of Foxl2. A myriad of antisense RNAs have been

identified so far, but many of them are described to display

patterns of expression dissimilar to their sense counterparts

[13]. They are thought to silence the sense transcript

expression via the formation of RNA–RNA duplexes that

are rapidly targeted for degradation by double-stranded

ribonucleases. Only the most abundant transcripts (either

sense or antisense) would persist in the cells, explaining the

distinct sense and antisense patterns of expression [13].

However, some antisense RNAs are coexpressed with the

sense transcripts and have no obvious silencing effect on

the expression of the resulting protein. This is the case with

EMX2 [17], WT1 [18], HIF-1a [19], N-myc [14], and, as

described here, Foxl2. In these instances, the antisense-

mediated regulation might be different from what has been

mentioned above and may even have a positive effect on

sense expression. The results of Moorwood and colleagues

[20] are in agreement with an antisense-mediated positive

effect: they have shown that WT1 antisense transcription

could increase WT1 protein level in vitro. Antisense

transcription may increase the accessibility of the chroma-

tin of the locus, leading to an increased expression of sense

transcripts [17,21]. Moreover, RNA duplexes may not be

systematically degraded and may stabilize the transcripts

involved. They may protect sense transcripts from degra-

dation. This has been recently observed in Chlamydomonas

chloroplasts, in which an antisense transcript has been

demonstrated to suppress instability of the polyadenylated

sense mRNA [22]. In addition, for the bcl-2/IgHfusion

locus, the antisense transcript has been proposed to protect

the sense transcript from degradation by masking part of

the bcl-2 3VUTR [13,23]. Foxl2OS transcripts may be

involved in similar mechanisms, to increase Foxl2tran-

scription or to stabilize its expression via RNA–RNA

duplex formation.

Up to now and despite the extreme conservation of

Foxl2 sequence and expression between human and

mouse, we have no evidence for the existence of an

antisense to FOXL2 transcript in human. Namely, prelimi-

nary RT-PCR experiments, similar to those described for

mouse Foxl2OS, have failed to produce any signal (data

no shown). Thorough experiments must be performed to

make a conclusion. However, the absence of a human

FOXL2OS would not be an exception. For instance, the

sequence and expression of WT1 are also very well

conserved between human and mouse, but antisense

transcription is detected only in human [18]. Instead of

an antisense transcript, the murine wt-1 locus possesses a

divergent transcript. Other experiments are needed to

determine if a similar phenomenon operates on the FOXL2

human locus.

In conclusion, we have identified new features of the

mouse Foxl2 locus. Foxl2 is expressed as two mRNA

isoforms resulting from an alternative polyadenylation. In

addition, antisense transcripts, named Foxl2OS, have been

shown to overlap the Foxl2 transcription unit. Foxl2 and

Foxl2OS are apparently coexpressed, even at the cellular

level. We suggest that the antisense may modulate Foxl2

expression via mechanisms that remain to be studied.

Table 2

List of primers

Name Sequence

Foxl2-mF 5V-GAACTAGAGCACTTTTGTTG-3VFoxl2-mTF 5V-AATGTCTTGTTTCTTCCACCT-3VFoxl2-mF2 5V-TCCCTTGTCAATTCCCAAAT-3VFoxl2 ORF-1R 5V-GTAGCTGGCCATCATGACAAA-3VFoxl2-5Vext reverse 5V-TGCAGGCGGGCGCAGGAGTGT-3VFoxl2-5Vint reverse 5V-GCTCGGCGCAGAGCCTCTAAC-3VFoxl2-mE 5V-AACTCCTACAACGGCCTGGG-3VFoxl2-3VF 5V-CCGCACCCTCACGCACATCAT-3VFoxl2-3Vext 5V-GCTACCTGGCGCCACCCAAGTA-3VFoxl2-short-R 5V-TTTTTTTTTTTTTTAAAAGAAAAACAAA-3VFoxl2-long-R 5V-TTTTTTTTTTTTTTCTGAAATCTGGA-3VFoxl2-6000R 5V-GTAGCACAGACTGACCTCAA-3VFoxl2sens-PE1 5V-FAM-TCTAACTTCTGCAGGCTGCGG-3VFoxl2OS-PE1 5V-FAM-GAGCACTTTTGTTGTGTTTGTACG-3VFoxl2OS-2F 5V-CAAGTGTCACGCGTGGAA-3VFoxl2OS-2R 5V-ATTCATTCAGACCATTATCC-3VFoxl2OS-3R 5V-TCCTTTCCCTTCATCTCAAA-3VB2MF 5V-AGCCGAACATACTGAACTGCTACG-3VB2MR 5V-CGGCCATACTGTCATGCTTAACTC-3VG3PDH-F 5V-ACCACAGTCCATGCCATCAC-3VG3PDH-R 5V-TCCACCACCCTGTTGCTGTA3V


Materials and methods

Cell culture

L929 and AT29C cells [10] were grown in DMEM

supplemented with 10% fetal calf serum (Invitrogen),

penicillin (100 U/ml), and streptomycin (100 Ag/ml) in a

5% CO2 atmosphere at 378C. For FISH experiments cells

were grown on glass slides for 24 h.

RNA extraction

Total RNA were extracted from a pool of mouse male or

female gonads of several stages (11.5, 12.5, and 16 dpc and

5 and 40 dpp) using Trizol (Invitrogen). RNAs from ovary,

eyelid, liver, lung, skeletal muscle, kidney, and intestine of

adult female mice were extracted using the RNeasy kit

according to the manufacturerTs instructions (Invitrogen).

Poly(A)+ RNA was extracted from 1 � 107 AT29C cells

using the Fast Track 2.0 kit (Invitrogen).

Northern blot

Ten micrograms of total ovary RNA and 8 Ag of AT29C

poly(A)+ RNAwere denatured for 10 min at 688C and run in

a 0.8% agarose gel containing 2 mol/L formaldehyde in 10

mmol/L phosphate buffer, pH 7. RNAs were then trans-

ferred onto Hybond-N+ membranes (Amersham) and cross-

linked by UV irradiation. Prehybridization and hybridization

were performed in ExpressHyb buffer (Clontech) according

to the manufacturerTs instructions. As probe we used a PCR

amplicon containing the first 381 bp of the Foxl2 ORF

(primers pBAD-FOXL2-F and FOXL2-B, previously

described in [1,2]). After gel purification (Qiaquick Gel

Extraction Kit, Qiagen), this PCR product was radiolabeled

by random priming using the NEBlot kit (New England

Biolabs) using [a-32P]dCTP (Amersham) and purified on

Nick Columns (Amersham). To detect Foxl2OS, we

performed an asymmetric PCR and obtained a single-

stranded probe that corresponds to the same 381-bp region.

To perform asymmetric PCR, pBAD-FOXL2-F primer was

diluted 1/100 with regard to the FOXL2-B. The resulting

product was processed as described above.

RACE

Foxl2 3VRACE. Reverse transcription was performed on 2

Ag of RNA from mouse ovary or AT29C cells with an

oligo(dT)-anchor primer following the instructions of the 5V/3VRACE kit (second generation; Roche Diagnostics). Two

heminested PCRs were then performed with Foxl2-mTF

primer and anchor primer for PCR 1 and Foxl2-mF2 and

anchor primer for PCR 2. Controls without cDNA or with

only one primer were performed in parallel. Two specific

bands were cloned and sequenced. Primer sequences are

shown in Table 2.

Foxl2 5VRACE. Reverse transcription was performed on 2

Ag of RNA from mouse ovary with a reverse primer located

at the beginning of the Foxl2 ORF (Foxl2 ORF-1R).

Purification and d(A)-tailing of the products were per-

formed following the manufacturerTs instructions (Roche

Diagnostics). Two nested PCRs were then performed with

Foxl2-5Vext reverse primer and oligo(dT) for PCR 1 and

Foxl2-5Vint reverse primer and anchor primer for PCR 2.

Controls without cDNA or with only one primer were

performed in parallel. Four specific bands were observed

and sequenced.

Foxl2OS 5VRACE. 5VRACE was performed as described

for Foxl2 with the exception that primer Foxl2-mTF was

used for reverse transcription. Two nested PCRs were

then carried out with Foxl2-mF and oligo(dT)-anchor

primers for PCR 1 and Foxl2-mF2 and anchor primers for

PCR 2. The resulting PCR products were cloned and

sequenced.

Foxl2OS 3VRACE. 3VRACE reverse transcription was

performed as described for Foxl2. Numerous PCRs were

then realized on the resulting cDNA with the anchor

primer and several gene-specific primers located from the

beginning of the Foxl2 transcriptional unit, to the region of

the putative antisense polyadenylation site, detected in the

EST database (located 9 kb downstream of the Foxl2OS

TSS).

Fluorescent primer extension

Fluorescent primer extension was carried out essentially

as described by Patek and colleagues [24]. A primer (0.14


Ag) carrying a 5Vfluorescent (6-FAM) label was annealed to

15 Ag of AT29C total RNA, in a final volume of 15 Al. ForFoxl2 primer extension, we used 5V-FAM-Foxl2sens-PE1

reverse primer (located at approx �104 bp with respect to

the first ATG codon of Foxl2). For Foxl2OS primer

extension, we used 5V-FAM-Foxl2OS-PE1 reverse primer

(located ~60 bp upstream of the Foxl2 first polyadenylation

site). Reverse transcription was then performed with Tran-

scriptor reverse transcriptase at 558C according to the

manufacturerTs instructions (Roche Diagnostics) and fol-

lowed by RNase A treatment. The resulting cDNA was

precipitated, resuspended in a mix of formamide/EDTA/

Genscan-2500 ROX (ABI fluorescent marker), denatured

for 3 min at 958C, and loaded onto a 6% polyacrylamide–

urea sequencing gel. Gels were run under standard electro-

phoresis conditions on an ABI 370A sequencer and the

electrophoregrams were analyzed using the GenScan soft-

ware. Controls were treated following the same protocol but

without reverse transcriptase.

RT-PCR

Reverse transcription. One microgram of total RNA was

treated with DNase I (Invitrogen). Reverse transcription was

carried out at 558C by Transcriptor reverse transcriptase

(Roche Diagnostics), with 33 ng of oligo(dT) and 3 ng of

primer Foxl2OS-3R (cf. Fig. 2B).

Foxl2-short and -long PCR. Two microliters of cDNA

products was amplified for 33 cycles with two pairs of

primers. For Foxl2-short, Foxl2-mTF and Foxl2-shortR

primers were used. For Foxl2-long, Foxl2-mF2 and Foxl2-

longR primers were used. Reverse primers were designed to

be specific for each isoform.

Foxl2OS PCR. Two microliters of cDNA products was

amplified for 35 cycles with a pair of primers that flanks

Foxl2OS exons 1, 2, 3, and 4 (Foxl2-ORF-1R and

Foxl2OS-2R primers) (Fig. 4A).

Two microliters of cDNA products was amplified for 35

cycles with a pair of primers that flanks Foxl2OS exons 3

and 4 (Foxl2OS-2F and Foxl2OS-2R primers) (Fig. 4D).

b2-Microglobulin PCR. Two microliters of cDNA products

was amplified for 30 cycles with B2MF and B2MR

primers.

Table 3

Conditions of semiquantitative fluorescent PCR

Foxl2-short Foxl2-long

Pair of primers mTF and shortR mF2 and lo

Annealing temperature 538C 538CElongation time (s) 20 20

Number of cycles 23 23

+/� DMSO 10% DMSO 10% DMSO

Size of the PCR product(s) 250 bp 450 bp

Fluorescence polymerase chain reaction

Two microliters of cDNA was amplified using the same

three pairs of primers described above (i.e., Foxl2-mTF and

Foxl2-shortR for Foxl2-short, Foxl2-mF2 and Foxl2-longR

for Foxl2-long, Foxl2OS-2F and Foxl2OS-2R for Fox-

l2OS), each forward primer being fluorescently labeled (6-

FAM). B2M amplification (with 5V-FAM-B2MF and B2MR

primers diluted 1/15 relative to the other primers) was

performed in duplex, as internal control. Following PCR, 1

Al of the resulting product was mixed with 4 Al of

formamide/EDTA/ROX and run as previously described

on an ABI 370A sequencer. Fluorescent PCR products were

quantified by measuring the areas of their corresponding

peaks in the electrophoregram. They were normalized with

respect to the internal B2Mcontrol and multiplied by 1000.

PCR conditions are described in Table 3.

RNA FISH

Single-stranded probe directed against Foxl2. A PCR

product corresponding to the first 381 nucleotides of Foxl2

ORF was amplified with primers pBAD-FOXL2F and

FOXL2-B [1,2] and cloned by Topo-TA cloning in the

pCR2.1 vector (Invitrogen). The resulting construction was

linearized by Hind III restriction enzyme (Hind III site

located ~60 bp downstream of the insert). The single-

stranded riboprobe directed against Foxl2 was produced by

in vitro transcription using T7 RNA polymerase and

digoxigenin–UTP (Roche Diagnostics; Riboprobe in vitro

transcription, Promega).

Single-stranded probe directed against G3PDH. A single-

stranded probe directed against G3PDH mRNAwas used as

a positive control. A G3PDH PCR fragment of 450 bp was

obtained by PCR with G3PDH-F and G3PDH-R primers

and processed as described above for the probe directed

against Foxl2.

Oligonucleotides directed against Foxl2OS. To detect

Foxl2OS, three 50-mer oligonucleotides carrying a 5Vbiotinmodification were purchased from Invitrogen. They were

designed to anneal to regions that are shared by Foxl2OS

variants (i.e., between the alternative exons) and not to

overlap the Foxl2 transcription unit. Sequence specificity

was assessed by BLAST against GenBank NCBI.

Foxl2OS B2M

ngR Foxl2OS-2F and 2R B2MF and B2MR

558C 53 or 558C40 20 or 40

25 23 to 25

No DMSO + or �572, 487, 404, and 319 bp 210 bp


The oligo sequences were as follows: Foxl2OS-Oligo1,

5V-BIO-CACGTGTGTCTTTAAGAACAGATTTTTGCCC-CGTGCCTTCCACGCGTGACACTTG-3V; Foxl2OS-

Oligo2, 5V-BIO-CTCAGCAACATCCTCGTCTGTGTC-

CAAGCCAGAGTACAAGGGATACCACTT-3V; Foxl2OS-Oligo3, 5V-BIO-CCTGAAAGCTGCCCAGAGGCTTG-

GATCACCTCTCCTCGCTGGGGTCAGCATTC-3V.

Irrelevant probe and oligonucleotides. A B2M PCR frag-

ment of 210 bp was obtained by PCR with B2M-F and B2M-

R primers and processed as described above. The resulting

single-stranded probe was chosen to anneal to hypothetical

B2M antisense transcripts. As no such transcripts have ever

been described, this was called an birrelevant probeQ and usedas a negative control. The irrelevant oligonucleotides

correspond to the antiparallel sequences of Foxl2OS-Oligo1,

2, and 3 and were used as negative controls.

Cells were fixed in 4% paraformaldehyde/phosphate-

buffered saline (PBS) for 15 min at room temperature. After

three washes for 5 min in PBS, cells were incubated 15 min

in PBS/1% Triton X-100. Cells were then dehydrated

through a series of ethanol solutions (70, 90, and 100%).

Hybridization was carried out with single-stranded probes in

25% deionized formamide/5� DenhardtTs/2� sodium

chloride–sodium citrate (SSC)/0.2 Ag/Al salmon sperm

DNA, overnight in a humidified chamber at 378C. Detectionwas carried out in 4� SSC/3% bovine serum albumin with

an anti-DIG FITC antibody (1/500; Roche Diagnostics) to

detect probes directed against Foxl2 or G3PDH or to detect

irrelevant probe. Detection was carried out in the same

buffer with streptavidin–Texas red (1/1000; Roche Diag-

nostics) to detect biotinylated oligonucleotides directed

against Foxl2OS or to detect irrelevant oligonucleotides.

Acknowledgments

The authors are grateful to Claire Francastel, Leila

Maouche-Chretien, and the other members of U567 for

assistance and advice. We thank the three anonymous

reviewers for their interesting comments. We also thank

Marie-Anne Ripoche and Eric Pailhoux for providing the

mouse tissue samples and Sandrine Caburet and Daniel

Vaiman for reading the manuscript. The AT29C cell line

was kindly provided by Nathalie di Clemente. J.C.,

R.A.V., and M.F. are funded by the Universite Paris 7,

the INSERM, and a GIS grant.

References

[1] J. Cocquet, et al., Evolution and expression of FOXL2, J. Med. Genet.

39 (2002) 916–921.

[2] L. Crisponi, et al., The putative forkhead transcription factor FOXL2

is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome,

Nat. Genet. 27 (2001) 159–166.

[3] E. De Baere, et al., Spectrum of FOXL2 gene mutations in

blepharophimosis–ptosis–epicanthus inversus (BPES) families dem-

onstrates a genotype–phenotype correlation, Hum. Mol. Genet. 10

(2001) 1591–1600.

[4] J. Cocquet, et al., Structure, evolution and expression of the FOXL2

transcription unit, Cytogenet. Genome Res. 101 (2003) 206–211.

[5] K.A. Loffler, D. Zarkower, P. Koopman, Etiology of ovarian failure in

blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a

conserved, early-acting gene in vertebrate ovarian development,

Endocrinology 144 (2003) 3237–3243.

[6] M.S. Govoroun, et al., Isolation of chicken homolog of the FOXL2

gene and comparison of its expression patterns with those of

aromatase during ovarian development, Dev. Dyn. 231 (2004)

859–870.

[7] D. Baron, et al., An evolutionary and functional outlook of FoxL2 in

the rainbow trout gonad differentiation, J. Mol. Endocrinol. 33 (2004)

705–715.

[8] D. Schmidt, et al., The murine winged-helix transcription factor Foxl2

is required for granulosa cell differentiation and ovary maintenance,

Development 131 (2004) 933–942.

[9] M. Uda, et al., Foxl2 disruption causes mouse ovarian failure by

pervasive blockage of follicle development, Hum. Mol. Genet. 13

(2004) 1171–1181.

[10] M. Dutertre, et al., Ovarian granulosa cell tumors express a functional

membrane receptor for anti-Mullerian hormone in transgenic mice,

Endocrinology 142 (2001) 4040–4046.

[11] G. Edwalds-Gilbert, K.L. Veraldi, C. Milcarek, Alternative poly(A)

site selection in complex transcription units: means to an end? Nucleic

Acids Res 25 (1997) 2547–2561.

[12] M. Kumar, G.G. Carmichael, Antisense RNA: function and fate of

duplex RNA in cells of higher eukaryotes, Microbiol. Mol. Biol. Rev.

62 (1998) 1415–1434.

[13] C. Vanhee-Brossollet, C. Vaquero, Do natural antisense transcripts

make sense in eukaryotes? Gene 211 (1998) 1–9.

[14] G.W. Krystal, B.C. Armstrong, J.F. Battey, N-myc mRNA forms an

RNA–RNA duplex with endogenous antisense transcripts, Mol. Cell.

Biol. 10 (1990) 4180–4191.

[15] D. Kimelman, M.W. Kirschner, An antisense mRNA directs the

covalent modification of the transcript encoding fibroblast growth

factor in Xenopus oocytes, Cell 59 (1989) 687–696.

[16] S. Nikic, D. Vaiman, Conserved patterns of gene expression in mice

and goats in the vicinity of the Polled Intersex Syndrome (PIS) locus,

Chromosome Res. 12 (2004) 465–474.

[17] F.C. Noonan, P.J. Goodfellow, L.J. Staloch, D.G. Mutch, T.C. Simon,

Antisense transcripts at the EMX2 locus in human and mouse,

Genomics 81 (2003) 58–66.

[18] Y. Gong, H. Eggert, C. Englert, The murine Wilms tumor suppressor

gene (wt1) locus, Gene 279 (2001) 119–126.

[19] F. Rossignol, et al., Natural antisense transcripts of HIF-1alpha are

conserved in rodents, Gene 15 (2004) 121–130.

[20] K. Moorwood, et al., Antisense WT1 transcription parallels sense

mRNA and protein expression in fetal kidney and can elevate protein

levels in vitro, J. Pathol. 185 (1998) 352–359.

[21] C. Bonifer, Long-distance chromatin mechanisms controlling tissue-

specific gene locus activation, Gene 238 (1999) 277–289.

[22] Y. Nishimura, E.A. Kikis, S.L. Zimmer, Y. Komine, D.B. Stern,

Antisense transcript and RNA processing alterations suppress

instability of polyadenylated mRNA in Chlamydomonas chloroplasts,

Plant Cell 16 (2004) 2849–2869.

[23] S. Capaccioli, et al., A bcl-2/IgH antisense transcript deregulates bcl-2

gene expression in human follicular lymphoma t(14;18) cell lines,

Oncogene 13 (1996) 105–115.

[24] M. Patek, G. Muth, W. Wohlleben, Function of Corynebacterium

glutamicum promoters in Escherichia coli. Streptomyces lividans, and

Bacillus subtilis, J. Biotechnol. 104 (2003) 325–334.

Sense and antisense Foxl2 transcripts in mouse

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