RNA UK 2008
Role of the C-terminal domain of RNA polymerase
II in expression of small nuclear RNA genes
Sylvain Egloff1 and Shona Murphy
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
Abstract
Pol II (RNA polymerase II) transcribes the genes encoding proteins and non-coding snRNAs (small nuclear
RNAs). The largest subunit of Pol II contains a distinctive CTD (C-terminal domain) comprising a repetitive
heptad amino acid sequence, Tyr1 -Ser2 -Pro3 -Thr4 -Ser5 -Pro6 -Ser7 . This domain is now known to play a major
role in the processes of transcription and co-transcriptional RNA processing in expression of both snRNA
and protein-coding genes. The heptapeptide repeat unit can be extensively modified in vivo and covalent
modifications of the CTD during the transcription cycle result in the ordered recruitment of RNA-processing
factors. The most studied modifications are the phosphorylation of the serine residues in position 2 and 5
(Ser2 and Ser5 ), which play an important role in the co-transcriptional processing of both mRNA and snRNA.
An additional, recently identified CTD modification, phosphorylation of the serine residue in position 7 (Ser7 )
of the heptapeptide, is however specifically required for expression of snRNA genes. These findings provide
interesting insights into the control of gene-specific Pol II function.
Introduction
snRNA (small nuclear RNA) genes have specialized
TATA-less promoters comprising PSE (proximal sequence
element) and DSE (distal sequence element) [1]. The PSE
recruits the snRNA-specific transcription factor PTF (PSE
transcription factor)/PBP (PSE-binding protein)/SNAPc
(snRNA-activating protein complex) to the snRNA gene
promoter, which is a critical step for specific initiation.
In contrast with mRNA, snRNAs are neither spliced nor
polyadenylated. Instead of the polyadenylation signal found
in protein-coding genes, snRNA genes contain a conserved
3 -box element, located downstream of the snRNA-encoding
region [2]. This element directs the formation of pre-snRNAs
that go on to be further processed post-transcriptionally. The
3 -box is recognized by Integrator, another snRNA-specific
multi-subunit complex recruited specifically to snRNA genes
and required for the generation of the 3 formation of presnRNAs [3].
Transcription of all genes by Pol II (RNA polymerase
II) involves assembly of the pre-initiation complex, transcription initiation, elongation and termination. In common
with pre-mRNAs, newly transcribed pre-snRNAs are cotranscriptionally capped at the 5 -end [4]. Transcription
and RNA-processing reactions have been found to be
closely linked in vivo [5]. At the heart of this lies the
repetitive CTD (C-terminal domain) of Pol II largest subunit,
which serves to recruit and interact with many processing
factors [5]. The CTD comprises tandem repeats of the
heptapeptide sequence Tyr1 -Ser2 -Pro3 -Thr4 -Ser5 -Pro6 -Ser7 .
During transcription of protein-coding genes, recruitment
of processing factors by the Pol II CTD is closely linked
to its position along the gene and the phosphorylation state
of specific serine residues. Phosphorylation of Ser5 and Ser2
residues has been extensively studied and underpins the differential recruitment of specific processing factors to the
proximal and distal regions of genes. Such a correlation
between the phosphorylation state of the CTD and gene
location has not been made for snRNA gene transcription,
largely because the transcription unit is very short. Recently,
phosphorylation of the serine residue in position 7 (Ser7 ) of
the CTD heptapeptide has been reported [6]. Most studies
on phosphorylation of the CTD are based on the ChIP
(chromatin immunoprecipitation) assay using antibodies that
selectively recognize Ser2 , Ser5 and Ser7 phosphorylation
and/or the use of drugs that inhibit CTDK (CTD kinase)
activity [5,7]. We have tested the requirement for each
of the three serine residues of the CTD heptapeptide for
expression of snRNA genes by introducing mutations into
consensus repeats using a CTD complementation system
[8]. A comparison of the effect of these mutations on
expression of both snRNA and mRNA genes indicates that
phosphorylation of Ser5 activates capping of the 5 -ends of
transcripts and phosphorylation of Ser2 activates RNA 3 end formation in expression of both the gene classes. In
contrast, phosphorylation of Ser7 plays a gene-specific role
in the recruitment of Integrator exclusively to snRNA genes
(Figure 1).
Ser5 phosphorylation
Key words: C-terminal domain (CTD), cyclin-dependent kinase (CDK), RNA polymerase II, RNA
processing, small nuclear RNA (snRNA), transcription.
Abbreviations used: CDK, cyclin-dependent kinase; CTD, C-terminal domain; CTDK, CTD kinase;
Pol II, RNA polymerase II; PSE, proximal sequence element; snRNA, small nuclear RNA.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2008) 36, 537–539; doi:10.1042/BST0360537
Phosphorylation of Ser5 residues by CDK7 (cyclindependent kinase 7; Kin28 in yeast), a component of the
general transcription factor TFIIH (transcription factor
IIH), occurs near the 5 -end of protein-coding genes [9].
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Biochemical Society Transactions (2008) Volume 36, part 3
Figure 1 Role of the modifications of the Pol II CTD during transcription of snRNA genes
Phosphorylation of Ser2 , Ser5 and Ser7 is indicated by an encircled ‘P’. After recruitment of Pol II on to the promoter of snRNA
genes, the CTD becomes phosphorylated on Ser2 , Ser5 and Ser7 . Ser7 phosphorylation is required for efficient transcription
and the recruitment of Integrator. During transcription, Ser5 phosphorylation activates capping of nascent RNAs. Finally,
phosphorylation of both Ser2 and Ser7 is required for recognition of the 3 -box by the Integrator complex in order to direct
efficient 3 -end formation.
Ser5 -phosphorylated CTD is a landing pad that recruits and
activates the capping enzyme, the enzyme responsible for
the addition of the cap structure at the 5 -end of newly
synthesized RNAs [10]. Accordingly, Ser5 phosphorylation
is required for recruitment of the capping enzyme to sites
of transcription in vivo [9,11]. Like mRNAs, snRNAs are
capped [4] and Ser5 phosphorylation has been detected on
snRNA genes [7], where it is likely to play the same role
that is does in protein-coding genes. In fact, mutation of Ser5
residues to the non-phosphoacceptor alanine residue within
the CTD has the same effect on both types of gene, with a
drastic reduction in the steady-state level of RNAs [8]. This
probably reflects the requirement for Ser5 phosphorylation to
protect the 5 -end of mRNAs and snRNAs against trimming
by exonucleases. Thus Ser5 phosphorylation is likely to be
universally linked to capping of the 5 -end of transcripts in
expression of mammalian Pol II-dependent genes.
Ser2 phosphorylation
Phosphorylation on Ser2 is directed by CDK9 (CTDK-I in
yeast), the catalytic subunit of the P-TEFb (positive transcription elongation factor b) complex [12]. This phosphorylation is at its highest on the processively elongating
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Authors Journal compilation form of Pol II near the 3 -end of protein-coding genes
[9] and has been demonstrated to promote elongation,
efficient splicing and 3 -end processing [5]. Accordingly,
some elongation factors, splicing factors and polyadenylation
factors have been shown to associate only with the elongating
form of Pol II. Pol II transcribing the snRNA genes is
also phosphorylated on Ser2 , but in contrast with proteincoding genes, this phosphorylation event is not required for
elongation of transcription [7]. Introduction of an alanine
residue in position 2 of the CTD heptapeptide leads to a
defect in 3 -end processing for both types of gene [7,8].
This finding suggests that Ser2 phosphorylation can play
a fundamental role in distinct 3 -end processing reactions
independent of a role in elongation. Some protein-coding
genes (the replication-activated histone H2b and p21 genes)
can also be efficiently transcribed in the absence of Ser2
phosphorylation [7,13], and in the case of histone H2b genes,
Ser2 phosphorylation is not required for processing either [7].
Ser7 phosphorylation
Ser7 phosphorylation has recently been discovered but
the Ser7 kinase remains to be identified. Ser7 phosphorylation
reaches a peak towards the 3 -end of protein-coding genes [6],
RNA UK 2008
and can be detected on snRNA genes [8]. Interestingly, while
replacement of Ser7 by an alanine residue does not seem to
affect mRNA production, it drastically affects the level and
the 3 -end processing of snRNAs [8]. Phosphorylation of
Ser7 residues is critical for association of the snRNA-specific
Integrator complex with the CTD in vitro and mutation
of Ser7 abrogates Integrator recruitment to snRNA genes
in vivo. Because Ser2 phosphorylation is also required for
efficient 3 -end processing of snRNA, it is possible that a
combination of Ser2 and Ser7 phosphorylations specifies the
recruitment of the Integrator complex into snRNA genes.
Interestingly, mutation of Ser7 also reduces the transcription
of snRNA genes, suggesting that the Integrator complex
may be required for efficient transcription from snRNA
promoters [8]. The Integrator may therefore share some
properties with the Mediator complex, which is required for
efficient transcription of protein-coding genes.
Concluding remarks
snRNA genes have provided useful model systems to study
fundamental mechanisms of transcription by Pol II for the
last two decades [1]. The most recent studies aimed at
understanding the role of the CTD in snRNA expression
have revealed some interesting insights into gene-specific
transcription by Pol II [7,8,14,15]. The results of these
studies highlight an snRNA gene-specific requirement for
a residue within the CTD heptapeptide, and emphasize that
phosphorylation of Ser2 does not play a role in elongation of
transcription of all genes [7,8].
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Received 17 January 2008
doi:10.1042/BST0360537
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