Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
e-mail: neugebauer{at}mpi-cbg.de
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Summary |
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Key words: RNA polymerase II, Pre-mRNA processing, Transcription unit, Pre-mRNA splicing, Polyadenylation, 5' end capping of mRNA, Nuclear export
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Introduction |
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What goes on at transcription units (TUs)? From the point of view of Pol
II, the transcription process includes pre-initiation complex formation,
transcription initiation, elongation, termination and dissociation of Pol II
from the DNA template (Fig. 1).
From the point of view of the transcript, pre-mRNA processing includes five
processes: (1) 5' end capping, in which the 5' triphosphate of the
pre-mRNA is cleaved and a guanosine monophosphate is added and subsequently
methylated to produce m7GpppN; (2) editing, in which individual RNA residues
are converted to alternative bases (e.g. adenosine is converted to inosine by
base deamination) to produce mRNAs encoding distinct protein products; (3)
splicing, in which introns are removed and exons are ligated together by the
spliceosome; (4) 3' end formation, which involves pre-mRNA cleavage and
synthesis of the poly(A) tail; and paradoxically (5) degradation. A priori,
each of these modifications might occur independently of the others, since
most can occur in in vitro reconstituted systems on purified pre-mRNA
substrates. However, many studies have revealed functional relationships
between these processes and each (with the exception of editing) has been
shown to be co-transcriptional at least some of the time. Importantly, a
number of trans-acting factors required for pre-mRNA processing directly bind
to Pol II, which stimulates processing, and, in some cases, processing feeds
back to Pol II activity. This has led to the proposal that transcription and
processing occur in a `gene expression factory' composed of machines linked
together for the purposes of efficiency and regulation
(Bentley, 2002;
Cook, 1999
;
Maniatis and Reed, 2002
;
Proudfoot et al., 2002
).
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5' end capping: coupling is key |
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The 5' cap modification itself renders pre-mRNA and mRNA resistant to
the action of 5' to 3' exonucleases. In addition, the cap serves
as a binding site for two important factors: the cap-binding complex (CBC) in
the nucleus and the translation initiation factor eIF4E in the cytoplasm
(Lewis and Izaurralde, 1997).
Like capping, CBC binding is co-transcriptional
(Visa et al., 1996
), but there
is no evidence to date that recruitment of CBC to the cap requires any
specific coupling to the transcription machinery. CBC is composed of two
subunits, CBP80 and CBP20, and plays a role in splicing of the first intron
(Colot et al., 1996
;
Lewis et al., 1996a
;
Lewis et al., 1996b
), promotes
the nucleocytoplasmic export of U snRNAs
(Gorlich et al., 1996
) and
supports a `pioneer round' of mRNA translation in the cytoplasm before CBC is
exchanged for eIF4E (Fortes et al.,
2000
; Ishigaki et al.,
2001
). Thus, the rapid and highly specific addition of the
5' cap to Pol-II-transcribed RNAs has important consequences for the
lifetime of the (pre)-mRNA, and this cascade of events can be attributed to
the initial interaction of the capping enzymes with Pol II.
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Pre-mRNA splicing: a race against transcription time? |
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Understanding how splicing is integrated with transcription is more
complicated than understanding capping, because metazoan genes contain
multiple introns (an average of nine per gene in humans), which cannot serve
as splicing substrates until both the 5' and 3' ends of each
intron are synthesized. Thus, the time that it takes for Pol II to synthesize
each intron defines a minimal time and distance along the gene in which
splicing factors can be recruited and spliceosomes formed. The time that it
takes for Pol II to reach the end of the TU defines the maximal time in which
splicing could occur cotranscriptionally. In general, Pol II moves along the
DNA template at a rate of 1-1.5 kb/minute. In humans, introns (which average
3,300 bp) are ten times longer than exons (which average 300 bp)
(Lander et al., 2001). This
corresponds to a
3 minute transcription time for introns and only
30
seconds for exons. RNP formation at 3' splice sites in
Drosophila is observed 48 seconds after 3' splice site
synthesis, with intron removal occurring 3 minutes later
(Beyer and Osheim, 1988
). If
these rates are similar in humans, then by the time the 3' splice site
is recognized, the next exon may already be finished, and by the time splicing
could occur 3 minutes later the next intron will have been completed. This
opens up the possibility for competition among splice sites in alternative
splicing. Indeed, intron removal does not always occur in the order of intron
synthesis, which indicates that some splicing events occur much more rapidly
than others and that slower splicing events may occur post-transcriptionally
in the nucleoplasm (LeMaire and Thummel,
1990
; Wetterberg et al.,
1996
). Evidence for the interplay between transcription and
splicing kinetics comes from experiments in humans and yeast, in which changes
in transcription rate by introduction of transcriptional pause sites or the
mutation of elongation factors result in the alternative selection of splice
sites (Roberts et al., 1998
)
(Howe and Ares, personal communication). Moreover, exons upstream of
exceptionally long (>20 kb) introns are preferentially trans-spliced to
3' ss-exon RNAs expressed from a heterologous Pol II promotes in human
cells (Kikumori et al., 2002
)
showing that competition occurs within and is influenced by the time-frame of
transcript synthesis. The demonstration that transcriptional activators
influence alternative splicing by modulating Pol II elongation rates
(Kadener et al., 2001
)
provides a physiological relevance for this kinetic relationship and suggests
that alternative splicing in vivo may in part be due to transcriptional rather
than splicing regulation per se. It will be interesting to learn whether
members of an increasing number of trans-acting elongation factors also
regulate splice site choice by a similar mechanism. Undoubtedly, one parameter
of this type of regulation is the amount of time the nascent RNA has to bind
to trans-acting splicing factors before the next binding site or splice site
is made.
In addition to this kinetic link between transcription and splicing, there
is the distinct possibility that a physical link also exists. The pivotal
observation is that the Pol II CTD stimulates splicing in human cells
independently of its effects on capping or 3' end formation
(Fong and Bentley, 2001).
Addition of Pol II or the CTD alone also stimulates splicing in vitro
(Hirose et al., 1999
;
Zeng and Berget, 2000
), but
the molecular mechanism underlying this stimulation is unknown. Although the
search for such a link has focused on a proposed role for the CTD in directly
binding to splicing factors (Corden,
1990
; Greenleaf,
1993
), to date the only bona fide splicing factor shown to bind to
the CTD in vitro is the yeast U1snRNP component Prp40p, which has no known
homologue in metazoans (Morris and
Greenleaf, 2000
). Although a search for direct binding partners of
the CTD revealed a set of proteins containing arginine-rich domains similar to
those present in non-snRNP splicing factors, note that splicing factors that
have demonstrated splicing activity were not detected in those assays
(Yuryev et al., 1996
). Within
the Balbiani Ring genes, snRNPs are concentrated in intron-rich regions and
are relatively scarce in regions lacking introns
(Kiseleva et al., 1994
), which
suggests that splicing factors do not travel with Pol II within the TU. It is
important to note that, in contrast to capping, splicing of at least some
pre-mRNAs in fission and budding yeast can occur efficiently following
synthesis by RNA polymerase III (Pol III)
(Kohrer et al., 1990
;
Tani and Ohshima, 1991
), T7
RNA polymerase (Dower and Rosbash,
2002
) or a CTD-less Pol II (Licatosi, 2002). Therefore, the
stimulatory effect of the CTD on splicing may not be essential.
A recent study suggests that the effects of the CTD on splicing efficiency
are indirect and due to an interaction of splicing snRNPs with Pol II
elongation factors (Fong and Zhou,
2001). This study shows that snRNPs or the addition of an intron
to the transcription template stimulate Pol II elongation by the direct
binding of snRNPs to the elongation factor TAT-SF1; TAT-SF1 in turn binds to
P-TEFb, which phosphorylates the CTD and remains associated with it during
elongation (Fong and Zhou,
2001
). One implication of this finding is that Pol II elongation
machinery might bring snRNPs to active genes. This may explain the observation
by light microscopy that a gene transcribed by CTD-less Pol II fails to
accumulate snRNPs or members of the SR protein family of non-snRNP splicing
factors (Misteli and Spector,
1999
); however, because the nascent RNA produced by the CTD-less
pol II probably also lacks the 5' cap and CBC, this observation remains
open to other interpretations. Indeed, intronless genes transcribed by
wild-type Pol II fail to recruit SR proteins in similar assays, which suggests
that the nascent RNA plays an important role in splicing factor recruitment
(Huang and Spector, 1996
;
Jolly et al., 1999
).
Importantly, if the CTD were pre-loaded with snRNPs directly or indirectly
through P-TEFb/TAT-SF1, it would be difficult to understand how introns could
further increase the elongation rate. Taken together, the simplest explanation
for this set of observations is that recruitment of snRNPs and TAT-SF1 to TUs
is enhanced by the cooperative binding of snRNPs to splicing signals within
the nascent RNA and of TAT-SF1 to P-TEFb.
Despite the lack of evidence for direct binding of snRNP or non-snRNP
splicing factors to the CTD, prevailing models of transcription-splicing
coupling in the literature are based the assumption of binding
(Goldstrohm et al., 2001;
Maniatis and Reed, 2002
). The
underlying logic of the model is that the crystal structure of Pol II places
the CTD at the exit groove of Pol II from which the nascent RNA emerges
(Cramer et al., 2001
), and
placement of splicing factors at the outlet would promote their efficient
recruitment to cognate RNA-binding sites as the latter are made. However,
splicing factors such as snRNPs and SR proteins are present at quite high
concentrations in HeLa cell nuclei [1-10 µM for U1, U2, U4, U5 and U6
snRNPs (Yu, 1999), and 10-100 µM for the SR protein SF2
(Hanamura et al., 1998
;
Phair and Misteli, 2000
)]. The
affinity of at least one SR protein SRp55 for its binding site in the
alternatively spliced cTNT pre-mRNA is 60 nM
(Nagel et al., 1998
). Thus, a
compelling argument for why further concentration of splicing factors would be
advantageous has yet to be made. In particular, the observation that `small
exons must be recognized within a vast sea of introns'
(Maniatis and Reed, 2002
) does
not explain why splicing factors should be bound to the CTD, since the
introns, like the exons, would experience the same elevated concentration of
factors.
Additional open questions not addressed by the model include differences in
splicing rates between introns, differences in the order of intron removal,
and how alternative splicing could occur in the context of such
Pol-II-directed recruitment. Finally, it is unclear whether all of the
components of the spliceosome and/or every alternative splicing regulator
should be positioned at every actively transcribed gene or whether genes
accumulate factors differentially to reflect their particular biosynthetic
requirements. Interesting alternatives to generic splicing factor recruitment
by the CTD are provided by the findings that the SR protein family member SF2
binds directly to the transcriptional co-activator p52
(Ge et al., 1998) and that
alternative splicing can be influenced by promoter identity
(Cramer et al., 1999
;
Cramer et al., 1997
). Thus,
much more information regarding the molecular mechanisms of splicing factor
recruitment and spliceosome assembly is required before we will be able to
come to an understanding of co-transcriptional splicing that can either be
generalized to all genes or satisfyingly describe the differences among
genes.
Although coupling between transcription and splicing can be important, it
may be equally important for some transcripts that splicing continues
post-transcriptionally. The Drosophila Ubx pre-mRNA contains a 75 kb
intron that is recursively spliced: the first splicing event creates new
splice sites, which are subsequently recognized, and the transcript is spliced
again (Hatton et al., 1998).
This chain of events could occur co-transcriptionally, but a strict coupling
between splice-site synthesis and splicing factor binding must be ruled out. A
similar complication arises through RNA editing by the ADAR family of
adenosine deaminases, because editing sites occur at splice junctions where
intron sequences base pair with upstream exon sequences to produce a
characteristic stem loop (Keegan et al.,
2001
). By definition, this must occur before splicing, and indeed
editing can alter splice-site sequences to produce alternative splicing
(Rueter et al., 1999
). Thus,
depending on the site and kinetics of editing, splicing of edited transcripts
may be either co- or post-transcriptional. The proposal that alternative
splicing occurs more slowly than constitutive splicing and results in the
splicing of some introns post-transcriptionally
(Melcak and Raska, 1996
) is
supported by microscopic studies that have detected slow-splicing introns away
from the site of synthesis (Dirks et al.,
1995
; Johnson et al.,
2000
; Zachar et al.,
1993
). The movement of (pre)-mRNA away from the gene is not
thought to represent vectorial transport to the nuclear envelope, because the
rates and trajectories of mRNP movement are consistent with diffusion
(Melcak et al., 2000
;
Politz et al., 1998
;
Politz et al., 1999
;
Singh et al., 1999
;
Wilkie and Davis, 2001
);
rather, the diffusion of such transcripts to the envelope may provide
additional time for post-transcriptional splicing to occur.
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3' end formation: tied up with termination |
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In human and yeast cells, the CTD of Pol II contributes to the efficiency
of polyadenylation (Fong and Bentley,
2001; Licatosi, 2002), and the purified large subunit of Pol II
stimulates polyadenylation in vitro
(Hirose and Manley, 1998
).
There are many physical links between Pol II and the polyadenylation
machinery. Several components bind to the Pol II CTD (e.g. CPSF and
cleavage/polyadenylation factor IA) and to other components of the Pol II
holoenzyme (e.g. CPSF binds to TFIID, and CstF binds to the transcriptional
coactivator PC4) (Barilla et al.,
2001
; Calvo and Manley,
2001
; Dantonel et al.,
1997
; McCracken et al.,
1997
). Thus, extensive protein-protein interactions among the
polyadenylation factors themselves and with Pol II may help to coordinate
termination and polyadenlyation. In Chironomus, these two events are
temporally correlated (Bauren et al.,
1998
), and polyadenylation cleavage factors are required for
efficient termination in yeast (Birse et
al., 1998
). However, direct visualization of nascent transcripts
in Xenopus and Drosophila shows that cleavage often occurs
after the release of Pol II from the DNA
(Osheim et al., 1999
; Osheim,
2002), which suggests that a substantial fraction of polyadenylation occurs
post-transcriptionally. It is not known whether Pol II remains associated with
the mRNP as it is released from the TU; if it does, it might continue to
stimulate polyadenylation post-transcriptionally. Indeed, both hypo- and
hyper-phosphorylated forms of free Pol II are able to enhance polyadenylation
of a synthetic pre-mRNA substrate in nuclear extract
(Hirose and Manley, 1998
),
indicating that stimulation of polyadenylation by Pol II need not be
co-transcriptional.
In contrast to capping, polyadenylation is not solely specified by Pol II.
A small but significant set of Pol II transcripts, such as histone mRNAs,
snRNAs and snoRNAs, are not polyadenylated and undergo alternative mechanisms
of 3' end formation (for a review, see
Proudfoot et al., 2002);
likewise, rRNA, which is normally synthesized by RNA polymerase I (Pol I), is
not polyadenylated when synthesized by Pol II
(Nogi et al., 1991
). Thus,
polyadenylation targeting by Pol II can be overridden by other processing
signals. Indeed, polyadenylation signals in nascent yeast RNAs support partial
polyadenylation of mRNAs transcribed by either Pol I, T7 RNA polymerase or Pol
II lacking the CTD (Licatosi, 2002; Lo et
al., 1998
; McNeil et al.,
1998
; Dower and Rosbash,
2002
), which confirms that a strict coupling between Pol II and
polyadenylation is not required. Another case of modulation of polyadenylation
function occurs in alternative terminal exon usage, in which polyadenylation
sites in upstream exons are not used in favor of those sites found in
downstream alternative exons. This points to the importance of the strength of
the polyadenylation signals described above, which can determine the rate of
assembly of polyadenylation complexes on the nascent transcripts
(Chao et al., 1999
). Assembly
of polyadenylation complexes on alternative terminal exons or unpolyadenylated
transcripts may thus be relatively slow compared with the rates of splicing or
alternative 3' end formation. Thus, signals in the nascent RNA play a
defining role in where and whether the transcript is polyadenylated.
The interdependence of terminal intron splicing and polyadenylation
(Niwa et al., 1992) and their
temporal coincidence (Bauren et al.,
1998
) suggest a kinetic and/or physical link between the two
processes. The splicing factor U2AF65 binds to the polypyrimidine tract at all
3' splice sites, where it promotes annealing of the U2 snRNA with the
branchpoint. Interestingly, U2AF65 also binds to the C-terminus of PAP
(Vagner et al., 2000
), and
this additional binding interaction probably helps to define the terminal exon
for splicing and promotes the assembly of polyadenylation machinery within the
exon. The U1 snRNP binds at 5' splice sites and inhibits PAP, perhaps
suppressing premature polyadenylation/termination in long introns or before
the synthesis of the terminal exon
(Gunderson et al., 1997
). In
the case of alternative terminal exon usage in the IgM pre-mRNA, the kinetics
of polyadenylation probably play a role, since elevated levels of CstF-64 in
plasma cells promote the recognition of the weaker upstream polyadenylation
signal and preclude splicing to the downstream 3' splice site
(Takagaki and Manley, 1998
).
Conversely, the calcitonin/CGRP pre-mRNA undergoes alternative terminal exon
usage through the action of a splicing factor SRp20, which promotes splicing
and polyadenylation at upstream sites (Lou
et al., 1998
). These physical and kinetic links between
polyadenylation and splicing indicate that these two processes co-evolved.
Because polyadenylation is linked with termination, interactions with the
splicing machinery may, on the one hand, have put pressure on splicing to
occur co-transcriptionally and, on the other hand, may have selected for
splicing to occur slowly enough to permit assembly of downstream complexes on
polyadenylation sites that might be otherwise spliced out too quickly.
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Release of mRNPs from the transcription unit: a distinct step? |
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How do mutations in export factors result in retention of transcripts at
TUs? Several lines of evidence link mRNA transport with transcription. First,
pre-mRNA splicing deposits a set of proteins called the exon-junction complex
(EJC) on mRNA, and this complex promotes the nucleocytoplasmic transport of
the mRNP (reviewed in Reed and Hurt,
2002). Second, even in the absence of splicing, two nuclear export
factors in yeast, Yra1p and Sub2p, and their human counterparts, ALY and
UAP56, are recruited to TUs through direct binding to the THO transcription
elongation complex (Lei et al.,
2001
; Strasser et al.,
2002
). This evolutionarily conserved transcription/export complex
(TREX) is detectable throughout the TU
(Strasser et al., 2002
), and
Yra1p has been detected in downstream regions of the TU in a separate study
(Lei et al., 2001
). The
co-transcriptional binding of these factors to nascent RNA raises the
possibility of a feedback mechanism that is active at the TU. This link
between RNA processing, mRNP release and nuclear export is reminiscent of
previous studies in human cells, showing that transcripts that exhibit
defective splicing or polyadenylation are retained at the TU
(Custodio et al., 1999
;
Horowitz et al., 2002
). It
remains to be determined how transcripts are retained at TUs and whether mRNP
retention in humans depends on components of the nuclear exosome.
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Conclusions and perspectives |
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Acknowledgments |
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References |
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