The CTD Role in Cotranscriptional RNA Processing and Surveillance

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FEBS Letters 582 (2008) 1971–1976

Minireview The CTD role in cotranscriptional RNA processing and surveillance

Se´rgio F. de Almeida, Maria Carmo-Fonseca* Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal

Received 4 April 2008; revised 13 April 2008; accepted 14 April 2008

Available online 22 April 2008

Edited by Ulrike Kutay

proteins specific to each snRNP. The selection of specific splice Abstract In higher eukaryotes, the production of mature messenger RNA that exits the nucleus to be translated into protein sites (ss) on a particular pre-mRNA substrate relies on an intri- requires precise and extensive processing of the nascent tran- cate interplay involving the cooperative binding of trans-acting script. The processing steps include 50-end capping, splicing, splicing proteins to cis-acting sequence elements in the pre- and 30-end formation. Pre-mRNA processing is coupled to tran- mRNA. In mammals, an intron is defined by four short and scription by mechanisms that are not well understood but involve poorly conserved consensus sequences: the exon–intron junc- the carboxyl-terminal domain (CTD) of the largest subunit of tions (50ss and 30ss); the branch point sequence; and the poly- RNA polymerase II. This review focuses on recent findings that pyrimidine tract (Table 1). These sequences are recognized by provide novel insight into the role of the CTD in promoting RNA base pairing with the spliceosomal snRNAs (Fig. 1). In addi- processing and surveillance. tion, both exons and introns contain weak binding sites for a Ó 2008 Federation of European Biochemical Societies. Pub- multitude of splicing auxiliary and regulatory proteins [6,7]. lished by Elsevier B.V. All rights reserved. Unlike yeast genes, which contain few and short introns, an Keywords: Splicing; Transcription; CTD; RNA polymerase II; average human gene consists of eight exons of 120 nucleo- RNA surveillance tides separated by introns at least 10 times longer in length [8]. Accurate recognition of multiple small exons embedded in large introns is thought to rely on a mechanism that involves cross-exon pairing between the 50ss and 30ss [9]. According to the ‘‘exon definition’’ model proposed by Berget, an interior 1. Introduction exon is first recognized by the paired binding of Ul and U2 snRNPs and associated splicing factors to the 50ss and30ss, fol- The expression of human protein-coding genes is remark- lowed by the juxtaposition of neighboring exons in the correct ably complex. The initial primary transcripts synthesized by order. The model further assumes that processing of the last RNA polymerase II (pre-mRNAs) are extensively processed exon involves interaction between splicing components at the 0 before functional messenger RNAs exit the nucleus to be trans- 3 ss and the polyadenylation complex [10,11], whereas recogni- lated into protein in the cytoplasm. Pre-mRNA processing tion of the first exon in a pre-mRNA is thought to be mediated steps include the addition of a 7-methyl guanosine (m7G) by interactions of the nuclear cap-binding complex (CBC) with 0 cap at the 50-end, splicing of introns, and 30-end formation the spliceosome [12]. Thus, capping, splicing and 3 -end forma- by cleavage and polyadenylation (Table 1). tion are intimately interconnected. The vast majority of human pre-mRNAs contain up to 90% of non-coding sequence that must be spliced. Excision of introns with single nucleotide precision relies on the spliceosome 2. Functional pairing of 50ss and 30ss occurs early in spliceosome (Table 1), one of the largest and most elaborate macromolec- assembly ular machines in the cell [1–3]. The building blocks of the spliceosome are uridine-rich small nuclear RNAs (U snRNAs) The biochemical reactions of pre-mRNA splicing involve packaged as ribonucleoprotein particles (snRNPs) that func- two transesterification steps [13,14]. In the first, the 20-OH of tion in conjunction with over 200 distinct non-snRNP auxil- the branch point adenosine is the nucleophile that attacks iary proteins [4,5]. The major spliceosomal small nuclear the 50ss phosphate, resulting in a free 50 exon and a lariat inter- ribonucleoprotein particles are the Ul, U2, U5 and U4/U6 mediate (Fig. 2). In the second step, the 30-OH of the 50 exon snRNPs. Each snRNP consists of one or two U snRNAs attacks the 30ss, resulting in release of the intron and concom- (Ul, U2, U5 and U4/U6 snRNAs) bound by a protein complex itant ligation of the two exons. A fundamental pre-requisite for that comprises seven common Sm proteins and one or more these reactions is the correct juxtaposition of branch point, 50ss and 30ss nucleotides. Indeed, hydroxyl-radical probing, a tech- *Corresponding author. Fax: +351 21 7999412. nique used to investigate RNA neighborhoods, revealed that E-mail address: [email protected] (M. Carmo-Fonseca). the branch point and the 30ss are located within 20 A˚ of the 50ss in the early stage of spliceosome assembly [15] (Fig. Abbreviations: CTD, Carboxyl-terminal domain of the largest subunit 3). More recently, the same technique was used to show that of RNA Polymerase II; U snRNP, Uridine-rich small nuclear 0 ribonucleoprotein particle; ss, splice site; Py, pyrimidine; m7G, nucleotides at the 5 end of U2 snRNA are already positioned 7-methyl guanosine near Ul and the functional sites of the pre-mRNA at the

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.04.019 1972 S.F. de Almeida, M. Carmo-Fonseca / FEBS Letters 582 (2008) 1971–1976

Table 1 Glossary Pre-mRNA Precursor of mature messenger RNA, the primary transcript of a protein-coding gene that is synthesized by RNA polymerase II Exon Exons are the regions of the primary RNA transcript (or the DNA that encodes them) that are present in the mature messenger RNA, which exits the nucleus and undergoes translation in the cytoplasm to produce protein Intron The term intron derives from ‘‘intragenic region’’. Introns are non-coding sections of an RNA or corresponding gene that are not present in the mature messenger RNA. Introns are also called intervening sequences Pre-mRNA splicing The process by which introns are removed from the pre-mRNA and exons are ligated Spliceosome The macromolecular machine composed of RNA and protein that is required for pre-mRNA splicing U snRNPs Uridine-rich small nuclear ribonucleoprotein particles 50 and 30ss The 50 and 30 splice sites correspond to the two exon–intron junctions that mark the beginning and end of an intron. They are also called splice donor and splice acceptor sites, respectively Branch point During the splicing reaction, introns are removed as a lariat structure in which the 50 end of the intron is joined to an adenosine located 17–40 nucleotides upstream of the 30 splice site. This A residue is called the branch point because it forms a branch in the lariat structure Py-tract Pyrimidine (py)-rich region of approximately 15 nucleotides near the 30ss, downstream of the branch point A CTD Carboxyl-terminal domain of the largest subunit of RNA Polymerase II SR proteins SR proteins are members of the serine/arginine-rich (SR) family of splicing factors. These proteins bind to speciﬁc pre-mRNA sequences known as exonic splicing enhancers (ESEs) m7G cap The 50-end of eukaryotic pre-mRNAs is modiﬁed by addition of a 7-methyl guanosine cap. The cap is bound by the nuclear cap binding protein (CBP) Cleavage The 30-terminus of a pre-mRNA is generally determined by endonucleolytic cleavage of the primary transcript at a site situated several hundred bases downstream from a conserved signal sequence. In most cases, cleavage is coupled to polyadenylation Polyadenylation Polyadenylation is the covalent addition of a series of adenosine residues (the so-called poly(A) tail) to the free 30-end of the RNA at the cleavage site

Fig. 1. Interactions between pre-mRNA, UlsnRNP and U2 snRNP early in the splicing process. The 50 region of Ul snRNA within the Ul snRNP complex initially base-pairs with nucleotides at the 50 splice site (50ss). The U2 snRNA within the U2 snRNP complex base-pairs with a sequence that includes the branch point A, which is not base-paired. The polypyrimidine (Py) tract and 30 splice site (30ss) are also indicated.

earliest stage of spliceosome assembly [16]. The earliest detect- How the U2 snRNA holds steady in the position for its sub- able spliceosomal complex (the E complex) contains the Ul sequent base pairing with the branch point sequence while the snRNP, splicing factors U2AF65, U2AF35 and SF1, members U2 snRNP is still loosely associated with the E complex? What of the SR protein family and the U2 snRNP, which at this bridges the two ends of an intron before the stable association stage is loosely associated. Subsequently, the U2 snRNP forms of U2 snRNP to the pre-mRNA? Although there are no deﬁn- an ATP-dependent stable interaction with the intron. In part, itive answers to these questions, it is currently assumed a mul- this interaction involves base pairing of the U2 snRNA with titude of weak protein–RNA and protein–protein interactions the branch point sequence and the adenosine nucleophile is se- play a key role in the 3D architecture of a spliceosome assem- lected because it bulges out of from this duplex [17]. Following bled on pre-mRNA. Namely, the U2 snRNP may associate recruitment of the U4/U6.U5 tri-snRNP to the spliceosome, with the branch point region via RNA contacts established the U6 snRNA establishes base pairing interactions with the by its subunits SF3a and SF3b, and/or by protein–protein 50 end of the U2 snRNA, and the resulting U2/U6 snRNA interaction with U2AF65 bound to the nearby polypyrimide structure forms the catalytic center of the spliceosome. tract [18]. Moreover, members of the serine/arginine-rich According to the data from Donmez et al. [16], the catalytically (SR) family of splicing factors (see below) facilitate interac- important 50 terminal nucleotides of U2 snRNA are immedi- tions between proteins bound across the intron [19–23]. Addi- ately positioned in close proximity to the reactive sites in the tional evidence points to a role of the carboxyl-terminal pre-mRNA (Fig. 3). domain of the largest subunit of RNA polymerase II (CTD) S.F. de Almeida, M. Carmo-Fonseca / FEBS Letters 582 (2008) 1971–1976 1973

Fig. 2. The chemistry of pre-mRNA splicing. The splicing reaction consists of two consecutive transesterification (replacement of one phosphodiester linkage for another) events. The figure depicts the result of the first step: 50 splice site cleavage and lariat formation. In the second step, the 30-OH of the 50 exon attacks the phosphodiester bond (–p) at the 30ss, resulting in release of the intron and concomitant ligation of the two exons.

Fig. 3. Structural organization of the pre-mRNA at the earliest stage of spliceosome assembly. The functional sites of the pre-mRNA, i.e. the 50 splice site, branch point sequence, and 30 splice site, are in close spatial proximity. Bending of the polypyrimidine tract induced by interaction with U2AF brings the 30 splice site into juxtaposition with the branch point sequence. Both the 50 and 30 splice sites are close to the 50-end of the U2 snRNA, which later assembles with U6 snRNA forming the catalytic center of the spliceosome. in bringing together exon–intron junctions, as discussed in the major site of reversible pol II phosphorylation. Five out of following section. the seven consensus residues can be phosphorylated, but residues Ser2 and Ser5 are the main targets of phosphorylation. RNA polymerase II with hypophosphorylated CTD is re- 3. Cotranscriptional tethering: a clamp to hold adjoining exons? cruited to the preinitiation complex and generates short transcripts by non-processive transcription [27]. Phosphorylation Ample evidence from a series of biochemical and cytologi- of Ser5 residues occurs at or near the promoter and correlates cal experiments demonstrates that splicing is tightly coupled with transcription initiation and early elongation (promoter to transcription and at least some introns are excised while clearance), whereas Ser2 phosphorylation associates with effi- the nascent transcript is still associated with the gene (for a cient transcript elongation [28]. comprehensive recent review see [24]). Not only splicing, but Truncation of the CTD causes defects in splicing in vivo [29– also capping and 30-end formation are coupled to transcrip- 31] and purified pol II or the CTD itself activates splicing in tion. This is achieved by recruitment of essential processing vitro [32,33]. However, the CTD only stimulated the splicing factors to the CTD of the largest subunit of RNA pol II of pre-mRNAs that contained complete exons with both 30ss [25]. Unlike all other DNA-dependent RNA polymerases, and 50ss. This led to the speculation that the CTD may immo- eukaryotic pol II contains a unique CTD that is highly con- bilize consecutive exons, thereby facilitating their juxtaposition served through evolution, increasing in length and diversifying [33]. Direct experimental support to this model was recently re- in structure with the complexity of organisms [26]. In yeast, ported by Proudfoot and co-workers [34]. The authors investi- the CTD consists of 26–29 repeats of a conserved heptapep- gated the splicing of pre-mRNAs containing an intron tide with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5- engineered to be cotranscriptionally cleaved. Disrupting the Pro6 -Ser 7. The mammalian CTD has 52 repeats, of which continuity of the nascent RNA had no effect on splicing of 21 obey the conserved consensus but the remainder displays the exons that flanked the cleaved intron. These results imply a variety of substitutions mostly located in the C-terminal that newly transcribed consecutive exons are somehow teth- part. At the very C-terminus, the mammalian CTD further ered to the transcription complex until they are spliced to- comprises a specific 10 amino acid motif. The CTD is the gether [34]. 1974 S.F. de Almeida, M. Carmo-Fonseca / FEBS Letters 582 (2008) 1971–1976

4. Are SR proteins part of the molecular ties responsible for exon required to mediate the eﬀect of an SR protein on alternative tethering? exon inclusion [44].

Binding of processing factors to the CTD increases their concentration in the vicinity of the nascent transcript, facilitat- 5. Checkpoints in mRNA biogenesis ing a short ‘‘molecular hop’’ to the pre-mRNA as the target sequences emerge from the polymerase [35]. Many proteins have The modification of the 50 ends of nascent RNA polymerase been described to bind to the CTD, including regulators of II transcripts by the addition of a m7G cap is the first pre- transcription initiation, histone methyltransferases, the pre- mRNA processing event in all eukaryotic cells. The cap is mRNA capping enzyme guanylyltransferase and factors in- added when the transcript is 25 bases long, soon after its volved in pre-mRNA 30-end cleavage and polyadenylation 50 end emerges from the exit channel of the polymerase. The [36]. reaction is catalyzed by the capping enzyme bound to the In a recent proteomic analysis of immunopurified human phosphorylated CTD. It is believed that after initiation of RNA polymerase II, Reed and co-workers identified SR pro- transcription, while the CTD is phosphorylated at Ser-5, a neg- teins and all of the components of the Ul snRNP, but no other ative elongation factor (NELF) is recruited through binding to snRNPs or splicing factors [37]. Consistent with this result, DRB sensitivity-inducing factor, DSIF [45]. This causes the several previous studies had reported that a subset of the SR arrest of the transcription complex before it enters into pro- proteins show preferential binding to the phosphorylated ductive elongation, giving ample time for capping. Most inter- CTD [38–41]. SR proteins are a highly conserved family of estingly, the capping enzyme interacts with both Ser-5 essential splicing factors that share a bipartite structure com- phosphorylated CTD and DSIF, and its recruitment disables posed of an N-terminal RNA binding domain and a C-termi- NELF-mediated repression of transcription [46]. This suggests nal arginine–serine-rich domain. SR proteins bind to specific that the capping enzyme plays a critical checkpoint role during exonic sequence elements (ESEs) and play a key role in recog- promoter clearance [46]. More recent data provides further nizing splicing signals and promoting spliceosome assembly support to this ‘‘checkpoint model’’ ensuring that uncapped [22,42]. It is therefore conceivable that the Ul snRNP and transcripts are not elongated [47]. Guiguen and colleagues SR proteins pre-loaded onto the CTD [37] bind to the nascent showed that depletion of the fission yeast cap-methyltransfer- transcript as the50ss and ESE sequences emerge from the poly- ase compromised the activity of Cdk9-Pchl (the kinase com- merase (Fig. 4). Assuming that SR proteins bridge their exonic plex that favors CTD phosphorylation on Ser2), resulting in binding sites to the CTD, SR proteins may provide the molec- promoter-proximal pol II stalling [47]. ular ties that tether adjoining exons to RNA pol II as recently Another point of coordination in mRNA biogenesis is the reported by the Proudfoot laboratory [34]. Consistent with this formation of pre-mRNA 30-ends. The 30-end of all eukaryotic view, binding of SR proteins to ESEs was previously shown to mRNAs, with the exception of the replication-dependent play a critical role in ensuring the correct 50 to 30 linear order histone-encoding transcripts, consists of a tail of adenosine of exon joining during splicing [43], and the CTD was found residues. Poly(A) tails are added by a template-independent

Fig. 4. Binding of Ul snRNP and SR proteins to the CTD promotes rapid cotranscriptional spliceosome assembly. Recent data from the Reed laboratory indicate that Ul snRNP and SR proteins associate with RNA pol II, possibly to the CTD [37]. This may increase the fidelity of the earliest recognition of the 50 splice site by Ul snRNA, as well as the efficient recruitment of SR proteins to exonic splicing enhancers (ESEs). The bound SR proteins (depicted in red) may thus act as clamps that hold adjoining exons tethered to the polymerase [34]. S.F. de Almeida, M. Carmo-Fonseca / FEBS Letters 582 (2008) 1971–1976 1975 poly(A) polymerase following endonucleolytic cleavage at a keeping mRNA biogenesis under tight quality control is well site in the 30-end of the pre-mRNA that is specified by con- illustrated by the growing number of human diseases known served sequence elements [48]. The cleavage and polyadenyla- to be caused by errors in mRNA processing [7,60]. tion machinery consists of a number of proteins that together recognize the poly(A) site, cleave the transcript and add the poly(A) tail. Some of these proteins bind directly to the Acknowledgements: We thank our colleagues M. Campinho, J. Rino and H. Hallay for helpful discussions. Our laboratory is supported CTD, preferentially to repeats phosphorylated on Ser-2 by Grants from Fundacao para a Cieˆncia e Tecnologia, Portugal [49,50]. In mammals, as well as in yeast, defects in pre-mRNA (PTDC/SAU-GMG/69739/2006), and the European Commission 30-end formation cause accumulation of aberrant transcripts (LSHG-CT-2005-518238, EURASNET). S.F. de A. is supported by near the site of transcription [51,52]. It was therefore proposed a fellowship from Fundaçaõ para a Ciencia e Tecnologia (SFRH/ BPD/34679/2007). that release of a nascent transcript from the site of transcription may be a checkpoint for mRNA integrity [53]. Evidence for an involvement of the CTD on mRNA release from the References transcription site is provided by the finding that a truncated CTD containing 31 repeats (heptads 1–23, 36–38 and 48–52) [1] Nilsen, T.W. (2003) The spliceosome: the most complex macro- is sufficient to support transcription, splicing, cleavage and molecular machine in the cell? Bioessays 25, 1147–1149. polyadenylation. Yet, the resulting mRNAs are mostly re- [2] Jurica, M.S. and Moore, MJ. (2003) Pre-mRNA splicing: awash tained in the vicinity of the gene after transcriptional shutoff in a sea of proteins. Mol. Cell 12, 5–14. [3] Will, C.L. and Lu¨hrmann, R. (2005) Spliceosome structure and [54]. 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