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Interplay between chromatin and RNA processing
Olivier Mathieu1,2,3 and Nicolas Bouché4,5
The processing of pre-mRNAs, including the selection of
polyadenylation sites, is influenced by the surrounding
chromatin context. We review here recent studies in
Arabidopsis thaliana highlighting the intricate and reciprocal
interplay between chromatin state and RNA processing. The
studies have revealed that transcription can be influenced by
the presence, in gene introns, of combination of epigenetic
marks typical of heterochromatin. New factors binding to these
marks have been identified and shown to play key roles in
controlling the use of polyadenylation sites and processing of
functional mRNAs. Concomitantly, several proteins of both the
splicing and the polyadenylation machineries are also emerging
as regulators of DNA methylation patterns and chromatin
silencing.
Addresses
1
Clermont Université, Université Blaise Pascal, GReD, BP 10448,
F-63000 Clermont-Ferrand, France
2
CNRS, UMR 6293, GReD, F-63001 Clermont-Ferrand, France
3
INSERM, UMR 1103, GReD, F-63001 Clermont-Ferrand, France
4
INRA, UMR 1318, Institut Jean-Pierre Bourgin, RD10, F-78000
Versailles, France
5
AgroParisTech, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles,
France
Corresponding authors: Mathieu, Olivier ([email protected]) and Bouché, Nicolas ([email protected])
Current Opinion in Plant Biology 2014, 18:60–65
This review comes from a themed issue on Genome studies and
molecular genetics
Edited by Kirsten Bomblies and Olivier Loudet
1369-5266X/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.pbi.2014.02.006
Introduction
In eukaryotes, pre-mRNAs transcribed by the RNA Polymerase II (Pol II) are then processed in three additive
ways: addition of a 50 -7-methyl guanosine cap, 30 -end
polyadenylation, and intron splicing. Because these
pre-mRNA processing steps are initiated while Pol II
synthesizes the transcripts, they are certainly influenced
by the surrounding chromatin context. Integrating results
from various post-genomic approaches, including RNAseq that allows detecting splicing variants, and chromatin
immunoprecipitation and bisulfte sequencing allowing
determining landscape of epigenetic marks genome-wide, have greatly helped to reveal the extent of the interplay
between RNA processing and chromatin. Recent studies
in Arabidopsis point toward a role for chromatin in the
Current Opinion in Plant Biology 2014, 18:60–65
selection of polyadenylation sites; and reciprocally, several factors of both the splicing and the polyadenylation
machineries are emerging as regulators of chromatin
silencing.
Chromatin marks modulate the use of
polyadenylation signals
In plants, the Jumonji C (JmjC) domain-containing
protein INCREASE IN BONSAI METHYLATION1
(IBM1) is essential to prevent ectopic epigenetic modifications at protein-coding genes [1–3]. IBM1 is a
histone demethylase that specifically removes monomethylation and di-methylation of the lysine 9 of
histone H3 tails (H3K9me1,2). In ibm1 mutants, genes
not only accumulate H3K9me2 but also cytosine methylation at CHG (where H = A, T or C) sequence contexts. This occurs as the DNA methyltransferase
CHROMOMETHYLASE3 (CMT3) that methylates
CHG positions is recruited to chromatin through its
binding to H3K9me2 marks. In combination with
others, both H3K9me2 and CHG methylation are
two general hallmarks of highly silenced regions such
as transposons. The function of IBM1 in maintaining
genes free of H3K9me2 and CHG methylation is
essential for normal development and ibm1 mutants
show severe and pleiotropic phenotypes after only a
few generations. How the switch in epigenetic marks
occurring in ibm1 mutants impacts gene expression is
unclear. Heterochromatin-associated epigenetic marks
such as CHG DNA methylation and H3K9me2 can
potentially interfere with transcription and/or disturb
RNA processing; however, their presence is now shown
to be also required for proper RNA processing at loci
including the IBM1 gene itself.
The IBM1 gene encodes two different transcripts
(Figure 1). Only the longest one is functional and
can complement an ibm1 mutant [4]. The production
of full-length IBM1 mRNAs requires splicing of an
unusually large intron of about 2 kb located in the
middle of the gene, and which harbors an at least
700-bp long heterochromatin domain associated with
dense CG and CHG methylation and H3K9me2.
Mutants that impact CG (i.e. met1) or CHG methylation
(i.e. cmt3 or kyp) in this intronic region do not accumulate normal levels of long IBM1 transcripts, implying
that both types of DNA methylation are necessary to
correctly process the functional transcript. Consequently, downregulation of the functional IBM1
mRNA in met1 mutants [4] results in the ectopic
appearance of both H3K9me2 and CHG methylation
at several thousand genes [5,6].
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Interplay between chromatin and RNA processing Mathieu and Bouché 61
Figure 1
MET1
KYP
IBM1
CMT3
EDM2
BAH
IBM2
IBM1-L
RMM
(A)n
pre-mRNAs
IBM1-S
(A)n
mCG
mCHG
H3K9me2
polyadenylation signal
Current Opinion in Plant Biology
IBM1 expression is controlled by heterochromatic marks. Schematic representation of the INCREASE IN BONSAI METHYLATION1 (IBM1) locus
encoding a Jumonji C (JmjC) domain-containing histone H3K9 demethylase. The IBM1 gene contains a heterochromatin domain inside its large
seventh exon (gray). The DNA methyltransferases METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3) maintain CG and CHG
methylation, respectively, and H3K9me2 is catalyzed by KRYPTONITE/SUVH4 (KYP). CMT3 physically interacts with H3K9me2, while KYP binds
CHG-methylated cytosines thereby generating a self-reinforcing loop between DNA and histone methylation. The gene body of IBM1 also contains
cytosines methylated in the CG context like many actively transcribed Arabidopsis genes, although the precise role of this methylation remains to be
determined. The Bromo-Adjacent Homology (BAH) domain of IBM2 possibly interacts with intronic heterochromatic marks of IBM1, while the IBM2
RNA-Recognition Motif (RRM) domain might link to the Pol II-elongating pre-mRNAs and favor distal polyadenylation by preventing use of internal
intronic polyadenylation signal (star). IBM2 and ENHANCED DOWNY MILDEW2 (EDM2) might also promote transcription of IBM1 over the intronic
heterochromatin domain. Both IBM2 and EDM2 are required for the accumulation of the long form of IBM1 pre-mRNA IBM1-L encoding the JmjC
domain (in brown), whereas the short form of IBM1 pre-mRNA IBM1-S is predominant when IBM2 or EDM2 functions are compromised.
We [7] and others [8,9] found that mutants of the IBM2/
ASI1/SG1 gene, hereafter called IBM2, are morphologically very similar to ibm1 mutants, and the two mutants
share the same molecular phenotype with thousands of
genic regions invaded by heterochromatic silencing
marks. However, IBM1 and IBM2 have no particular
impact on transposable elements located in constitutive
heterochromatin. IBM2 encodes a protein of unknown
function containing a Bromo-Adjacent Homology (BAH)
domain shared by several chromatin regulators and an
RNA-Recognition Motif (RRM). In ibm2 mutants, as in
met1, the long intron of IBM1 is not correctly processed,
and consequently accumulation of full-length functional
IBM1 mRNAs is drastically compromised. This accounts
almost exclusively for the observed ibm2 phenotype as it
can be complemented by the introduction of a transgene
producing the long version of IBM1 transcripts. Complementation only occurs when the large intron was omitted
from the IBM1 transgene, implying that IBM2 functions
by promoting transcription of IBM1 over the intronic
heterochromatin domain in the wild-type (Figure 1).
IBM2 targets additional genes that contain long introns
associated with DNA methylation and chromatin immunoprecipitation assays confirmed that IBM2 physically
interacts with these regions. In the absence of IBM2,
mRNAs corresponding to these genes are shorter, precociously ending in their large introns because proximal
polyadenylation sites seem to be favored. This is raising
the possibility that IBM2 might function by promoting
the use of distal polyadenylation sites over proximal ones
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located in large introns. Using a new method allowing
sequencing of single mRNA molecules and direct identification of mRNA cleavage and polyadenylation sites, a
recent study has demonstrated that use of alternative
polyadenylation sites within introns of pre-mRNAs is
rare, FCA and FPA (see below) being two exceptions
[10]. Combined with the fact that large introns (>2 kb)
containing transposon-like sequences are also uncommon, at least in Arabidopsis [8], the role of IBM2 in
controlling polyadenylation is likely limited to few, very
specific loci. Accordingly, only four IBM2 target genes
have been characterized so far. First, IBM1 that controls
chromatin structure to ensure that highly transcribed
genes remain free of silencing marks. Second,
AT1G11270 that shows heat responsiveness due to the
insertion of a retrotransposon of the ONSEN family in its
second intron [11]. Third, a gene encoding a photosystem
II reaction center PsbP family protein. And finally, a
disease-resistance gene, RPP7, which contains a COPIA
retrotransposon in its first large intron. Although all these
genes contain intronic heterochromatin domains, their
nature is different, including a LINE element, two
COPIA retrotransposons, and in the case of IBM1, a
sequence homologous to both chloroplast and mitochondrial genes. Therefore, IBM2 might recognize its target
genes through a specific chromatin signature rather than a
precise DNA sequence.
Interestingly, polyadenylation of RPP7 is also controlled
by another protein, ENHANCED DOWNY MILDEW2
Current Opinion in Plant Biology 2014, 18:60–65
62 Genome studies and molecular genetics
(EDM2) that contains several zinc-finger domains and a
region similar to the active domains of methyltransferases
[12]. edm2 mutants were isolated from a genetic screen to
identify genes that are essential for RPP7 function [13].
Similar to ibm2, edm2 mutants show impaired accumulation of functional RPP7 transcripts. Levels of H3K9me2
at the intronic COPIA retrotransposon are reduced in
edm2 and this directly impacts the transcription of the fulllength RPP7 transcript [14]. In the wild-type,
H3K9me2 EDM2-dependent marks promote the use
of a distal polyadenylation site, thereby enhancing production of the long RPP7 functional transcript; while in
edm2 mutants, a proximal polyadenylation site localized in
the retrotransposon is used, leading to a shorter transcript
(named ECL). In natural Arabidopsis accessions containing an RPP7 gene with no COPIA elements, the alternative polyadenylation of the transcript becomes EDM2independent. The EDM2 protein can physically bind
H3K9me2 EDM2-dependent marks, and unlike IBM2,
EDM2 controls levels of H3K9me2 at certain transposons
[12]. The precise function of EDM2 remains to be
determined. Not surprisingly, EDM2 also binds heterochromatic marks of IBM1 and controls the expression of
the functional IBM1 long transcript by promoting the
distal polyadenylation site [15]. Consequently, the similar
phenotypic alterations observed in edm2, ibm1 and ibm2
mutants all result from the misregulation of IBM1.
A role for epigenetic marks in regulating alternative
polyadenylation has also been revealed in the unique
case of two mouse imprinted genes: H13 and Herc3 that
host in their introns two genes (called retrogenes) Mcts2
and Napl15, respectively, sharing characteristics with
retrotransposons [16,17]. When the retrogenes are
silenced by DNA methylation, a distal polyadenylation
site is used to process the host genes. In contrast, the
transcription of the unsilenced retrogenes favors a proximal polyadenylation site to the detriment of the host
gene. Experimental evidence points toward a mechanism
of direct competition for the transcription of the host and
the retrogene transcripts, the latter one being transcribed
and polyadenylated first when silencing marks are absent.
In the case of both IBM2 and EDM2, the mechanism
involved is co-transcriptional or post-transcriptional since
the Pol II occupancy of their target genes remains similar
between the wild-type and the corresponding ibm2 or
edm2 mutants [8,14,15].
RNA 30 -processing factors are required for
chromatin silencing
Plants control their flowering to ensure that they reproduce under favorable conditions. This complex control is
extensively studied in Arabidopsis, and studies of the
flowering repressor gene FLC have certainly been pioneer
in revealing a link between RNA processing activities and
chromatin regulation in gene silencing. Two RNA-binding proteins, FCA and FPA, are implicated in the
Current Opinion in Plant Biology 2014, 18:60–65
regulation of alternative polyadenylation, thereby playing
important roles in limiting intergenic transcription in the
Arabidopsis genome [18]. FCA not only regulates its own
expression by enhancing proximal polyadenylation but
also controls expression of the major floral regulator gene
FLC [19]. Together with FPA, FCA represses FLC
expression through alternative 30 -processing of the FLC
antisense transcript termed COOLAIR [20,21]. FCA and
FPA act to favor proximal polyadenylation of COOLAIR.
The resulting short FLC antisense RNA prompts transcriptional downregulation of FLC through stimulating
the activity of the FLD histone H3K4me1/2 demethylase
in an unknown process, leading to a repressed chromatin
state in the body of the FLC gene [19]. Such a role for
FCA and FPA in chromatin silencing is not restricted to
the FLC gene and appears to be more widespread than
may have been originally anticipated. Indeed, mutant
alleles of these two genes were recovered in a genetic
screen aimed at identifying further components required
for RNA-mediated chromatin silencing [22]. FCA and
FPA were revealed to be required for efficient transgene-induced chromatin silencing of the endogenous
PDS gene, as well as silencing of several, mainly
single-copy, endogenous sequences including some
transposable elements. At certain targets, transcriptional
reactivation in fca fpa double mutants was coupled to
reduced DNA methylation, mainly in non-CG sequence
contexts, suggesting for the first time a possible link
between 30 -RNA processing and DNA methylation.
RNA-directed DNA methylation and splicing
pathways are interconnected
The presence of non-CG methylation at a given DNA
sequence, and of CHH methylation in particular, is often
a signature of RNA-directed DNA methylation (RdDM)
[23]. The efficient maintenance of CHH methylation
requires the continuous production of 24-nt small interfering RNAs (siRNAs) that act as targeting molecules,
although a siRNA-independent CHH methylation pathway involving CMT2 has recently been reported in
Arabidopsis [24]. There is ample evidence that RdDM
contributes to gene silencing in plants, in particular at
transposable elements and other types of repeated DNA
sequences [25,26]. This complex pathway is likely
primed by the production of double-stranded RNAs
through the action of RNA polymerase IV and the
RNA-dependent RNA polymerase RDR2 [27]. The
resulting dsRNAs are then cleaved by DICER-LIKE3
(DCL3) into 24-nt siRNAs that are subsequently loaded
onto an ARGONAUTE protein (AGO4 or AGO6). siRNAs loaded onto AGO proteins then serve as sequence
specific guides for the DNA methyltransferase DRM2 by
pairing with nascent scaffold RNAs produced from target
loci by the RNA polymerase Pol V or Pol II [28,29]. Some
of the factors involved in RdDM, including RDR2, DCL3
and AGO4, as well as siRNAs were shown to colocalize
with nucleolus-associated organelles named Cajal bodies
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Interplay between chromatin and RNA processing Mathieu and Bouché 63
[30]. These bodies, described in 1903 by Ramón y Cajal,
appear to be universal nuclear organelles that have key
roles in nuclear physiology. Among those are processing
of ribosomal RNA and 30 -end histone mRNA, and the
modification and assembly of U small nucleolar ribonucleoproteins (snRNPs), some of which eventually form
the RNA splicing machinery also known as the spliceosome. Of the seven U snRNPs, only five are involved in
the spliceosome (U1, U2, U5 and the U4/U6 complex)
that is additionally composed of numerous non-snRNP
splicing factors [31,32]. The simultaneous presence of
RDR2, DCL3, AGO4 and siRNAs in Cajal bodies
suggests that these nuclear compartments also function
as centers for siRNA processing and AGO4 effector
complex assembly [30,33].
Therefore, given this spatial proximity, it may not sound
inappropriate to expect some type of functional relationship between RdDM and RNA splicing components. A
series of recent reports provide experimental support for a
role of RNA splicing factors in RdDM. The DRM2 DNA
methyltransferase involved in the RdDM pathway also
performs all de novo DNA methylation that occurs when
some naı̈ve/unmethylated DNA sequences are stably
introduced in the Arabidopsis genome [34]. Transformation of Arabidopsis with a transgene containing the FWA
gene has been extensively used as an assay for de novo
DNA methylation. Wild-type plants transformed with
FWA efficiently methylate repeats encompassing the
promoter of the FWA transgene leading to silencing,
whereas drm2 mutants do not initiate DNA methylation
allowing FWA expression, which induces a late flowering
phenotype. In an effort to identify additional factors
required for the establishment of DNA methylation,
Ausin and colleagues screened a collection of pre-selected
T-DNA insertion lines using the FWA transgene silencing reporter system [35]. A late flowering phenotype
was observed when plants mutant for ARGININE/SERINE-RICH45 (SR45) were transformed with the FWA
transgene. SR45 contains an RRM and was previously
shown to be a bona fide plant-specific splicing factor [36].
The sr45 mutant not only shows impaired de novo DNA
methylation capacity at the FWA transgene, but also
reduced non-CG methylation at several endogenous
RdDM targets, as well as impaired accumulation of
siRNAs associated with RdDM. Therefore, the SR45
splicing factor appears to be required for RdDM and is
probably acting in this pathway before the production of
siRNAs. Resembling FCA and FPA, SR45 was interestingly additionally shown to be required for proper silencing of FLC [35,36], suggesting that SR45 role in RNAmediated silencing extends beyond the RdDM pathway.
Last year, additional support for a role of the whole
mRNA splicing machinery in RdDM was provided by
studies from two other groups. Three mutants for
different splicing factors were recovered from forward
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genetic screens designed to identify suppressors of
RdDM-dependent silencing of a luciferase transgene.
Mutants were recovered in genes encoding ZINC-FINAND
OCRE
DOMAIN-CONTAINING
GER
PROTEIN1 (ZOP1), a novel pre-mRNA splicing factor
conserved in green algae and various angiosperms [37];
STABILIZED1 (STA1), a U5 snRNP-associated protein
required for both splicing and mRNA stability [38,39];
and RNA-DIRECTED DNA METHYLATION16
(RDM16), a component of the U4/U6 snRNP protein
complex conserved in eukaryotes [40]. Similar to sr45, all
three mutants show reduced non-CG methylation at
several endogenous RdDM targets, including AtSN1.
However, both the impact of these mutants on siRNA
accumulation and the nuclear localization of the corresponding wild-type proteins suggest that these splicing
factors act at different steps of the RdDM pathway.
Indeed, mutants for SR45, ZOP1 and STA1, but not
RDM16, affect siRNA abundance; whereas sta1 and
rdm16, but not zop1, reduces Pol V transcripts accumulation suggesting that STA1 and RDM16 likely function
in a later step of RdDM. ZOP1 can specifically associates
with STA1 [37], and the two factors colocalize with
AGO4 at the Cajal body [37,38], while RDM16 protein
is dispersed throughout the nucleoplasm [40]. In support
to a role of the whole splicing machinery in RdDM rather
than anecdotic contribution of particular components,
Zhang and colleagues showed that mutants of four other
spliceosome factors are also defective to various extents
for siRNA accumulation, DNA methylation and transcriptional silencing [37]. Importantly, all these
mutations do not significantly alter transcript accumulation and pre-mRNA splicing of known RdDM factors,
indicating that their contribution to RdDM and RNAmediated silencing is likely to be direct [37,38,40].
However, the functional contribution of the spliceosome
components into RdDM remains unclear. To this point,
no interactions between splicing factors and RdDM components have been detected. Besides, many RdDM targets with altered DNA methylation or transcriptional
silencing in mutants of these splicing factors are intronless sequences. Thus, it is plausible that it is more the
RNA processing activities of these factors than the premRNA splicing function itself that plays a role in RdDM.
Somehow similar to the function of FCA/FPA in processing non-coding FLC transcripts, the splicing factors may
interact with non-coding transcripts generated by either
Pol V or Pol II and process these co-transcriptionally so
they are able to act as scaffold RNAs in the later steps or
RdDM. Some of these spliceosome components such as
SR45 may also be required to process Pol IV transcripts
and allow them to be recognized as a template by RDR2
at early steps of RdDM. Alternatively, as sr45 shares a
very similar phenotype as dcl3 [35], and making the
parallel with the role of the core splicing factor SmD1 in
RNAi in Drosophila [41], SR45 may interact with DCL3
and/or dsRNAs to allow optimal siRNA biogenesis.
Current Opinion in Plant Biology 2014, 18:60–65
64 Genome studies and molecular genetics
Conclusions
Chromatin state and RNA processing meet at different
levels from which certainly only a few aspects are currently
known. The recent discovery of IBM2 and EDM2 raises
interesting questions related to mechanisms of interplay
between RNA polyadenylation and chromatin marks. The
mode of action of proteins linking pre-RNAs and chromatin is little understood and the discovery of protein partners
and complexes will certainly help resolve that issue. An
evolutionary common scheme in which pre-mRNA splicing factors modulate RNA-mediated silencing processes,
including RdDM is emerging from studies in fission yeast,
Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis [35,37,38,40,41–43]. Although it is likely that
these factors act in these processes independently of their
pre-mRNA splicing activities, the underlying molecular
mechanisms remain elusive. Isolation of additional
mutants through forward genetics and combination of
genomic and biochemical approaches will undoubtedly
soon provide a deeper understanding of the mode of action
of the splicing machinery in the RdDM pathway and in
RNA-mediated silencing in general. pre-mRNAs are processed while Pol II is still elongating transcripts, meaning
that splicing and alternative polyadenylation are potentially competing. The relationship between the rate of
elongation by the Pol II and pre-mRNA processing will
also need to be addressed in plants. In animals, the control
of alternative polyadenylation is emerging as an important
contributor to the complexity of transcriptomes [44]. The
discovery of underlying mechanisms of regulation in both
plants and animals is thus becoming an important research
priority.
Acknowledgements
We would like to thank Isabelle Gy for critical reading of the manuscript
and helpful comments. NB is supported by the ANR (Project 11-JSV70013). Work in OM’s laboratory is funded by the regional council of
Auvergne and by the European Community’s Seventh Framework
Programme (FP7 2007/2013) through a Starting Independent Researcher
grant from the ERC (I2ST-#260742). OM is an EMBO Young Investigator.
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Interplay between chromatin and RNA processing Mathieu and Bouché 65
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