Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses
2010; Springer Nature; Volume: 29; Issue: 13 Linguagem: Inglês
10.1038/emboj.2010.103
ISSN1460-2075
AutoresDmitry Cherny, Clare Gooding, Giles E. Eperon, Miguel B. Coelho, Clive R. Bagshaw, Christopher W. J. Smith, Ian C. Eperon,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle25 May 2010Open Access Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses Dmitry Cherny Dmitry Cherny Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Clare Gooding Clare Gooding Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Giles E Eperon Giles E Eperon Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Miguel B Coelho Miguel B Coelho Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Clive R Bagshaw Clive R Bagshaw Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Christopher W J Smith Christopher W J Smith Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Ian C Eperon Corresponding Author Ian C Eperon Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Dmitry Cherny Dmitry Cherny Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Clare Gooding Clare Gooding Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Giles E Eperon Giles E Eperon Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Miguel B Coelho Miguel B Coelho Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Clive R Bagshaw Clive R Bagshaw Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Christopher W J Smith Christopher W J Smith Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Ian C Eperon Corresponding Author Ian C Eperon Department of Biochemistry, University of Leicester, Leicester, UK Search for more papers by this author Author Information Dmitry Cherny1, Clare Gooding2, Giles E Eperon1, Miguel B Coelho2, Clive R Bagshaw1, Christopher W J Smith2 and Ian C Eperon 1 1Department of Biochemistry, University of Leicester, Leicester, UK 2Department of Biochemistry, University of Cambridge, Cambridge, UK *Corresponding author. Department of Biochemistry, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK. Tel.: +44 116 229 7012; Fax: +44 116 229 7018; E-mail: [email protected] The EMBO Journal (2010)29:2161-2172https://doi.org/10.1038/emboj.2010.103 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of α-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity. Introduction The scale of alternative splicing is breathtaking. It has been estimated on the basis of high-throughput sequencing of mRNA that there are about 100 000 significant alternative splicing events in humans, derived from about 90% of the multiexon genes; this corresponds to an average of seven events per multiexon gene (Pan et al, 2008; Wang et al, 2008). The actual number of alternative isoforms of mRNA depends on the combinations produced by each gene, but the number could exceed a million. Despite such overwhelming numbers, it seems that alternative splicing is not the result of errors (Pan et al, 2008) and that the error rate of splicing is so low that any errors may be in large part the result of misincorporation during transcription (Fox-Walsh and Hertel, 2009). Many, possibly most, alternative splicing events are represented more abundantly in particular tissues (Castle et al, 2008; Wang et al, 2008). The introns flanking tissue-specific exons are enriched in a number of sequence motifs, the most abundant of which contain UCUCU (Castle et al, 2008; Wang et al, 2008). This is recognized as a site, or partial site, for binding of polypyrimidine tract-binding protein (PTB) (Chan and Black, 1995, 1997). PTB was characterized as a PTB (Garcia-Blanco et al, 1989), and then identified as a possible repressor of a muscle-specific exon (Mulligan et al, 1992). Subsequently, it was shown to regulate the expression of a number of mRNA isoforms, particularly some specific to neural and muscle tissue (Grabowski and Black, 2001; Black and Grabowski, 2003; Boutz et al, 2007; Spellman et al, 2007; Venables et al, 2008). As the level of inclusion of exons with nearby intronic UCUCU motifs is inversely correlated among tissues with the abundance of PTB (Castle et al, 2008), repression by PTB seems to be a widespread and important contribution to tissue-specific gene expression. Indeed, genome-wide analysis of PTB–RNA association suggests that PTB is associated with transcripts from over 40% of annotated human genes (Xue et al, 2009). PTB has four RNA-binding domains of the RRM type, with flexible linkers after domains 1 and 2 (Petoukhov et al, 2006). There are three isoforms of PTB, which differ in the length of the linker between RRMs 2 and 3. PTB4 has the longest linker and is the most effective repressor of Tpm1 exon 3 (Wollerton et al, 2001). The individual RRMs bind RNA with low affinity and weak specificity for short pyrimidine tracts (Oberstrass et al, 2005). Structural analyses of the individual RRMs and of RRMs 3 and 4 together suggest that the first three RRMs could bind a consecutive sequence of at least 15 nucleotides but that the fourth RRM would require linking sequences before binding to a short pyrimidine tract (Oberstrass et al, 2005; Lamichhane et al, 2010). In contrast, the occluded binding site size on poly(U) for the intact protein was estimated to be 5 nts (Perez et al, 1997). The protein was found by selection experiments to recognize a pyrimidine-rich consensus of 26 nts (Singh et al, 1995), although experiments with natural substrates identified shorter high-affinity motifs of UCUCUCU (Chan and Black, 1997) or UCUU (Perez et al, 1997). In the absence of other proteins, PTB binds to RNA with canonical motifs to form small complexes with nanomolar affinity, and then larger complexes non-cooperatively (Singh et al, 1995; Amir-Ahmady et al, 2005; Clerte and Hall, 2006). The numbers of PTB molecules in the larger complexes were hard to predict but seemed to correlate more with the overall length of the pyrimidine tract than with specific motifs. An analysis of genome-wide binding sites suggested that the level of pyrimidines remains elevated over tens of nucleotides around each site (Xue et al, 2009). The mechanisms by which PTB association represses splicing are unclear. In several cases, pyrimidine-rich tracts are located on both sides of the regulated exon or splice site (Wagner and Garcia-Blanco, 2001; Amir-Ahmady et al, 2005). In the case of the neural exon of the Src gene, which is repressed in most tissues by the binding of PTB, two separate pyrimidine tracts on either side of the exon cooperate to form an ATP-resistant complex containing unknown numbers of proteins (Chou et al, 2000). The effect of this is to prevent the interaction of U1 snRNPs, bound to the 5′ splice site of the exon, with components at the downstream 3′ splice site (Sharma et al, 2005, 2008), but the nature of the impediment is unknown. Pyrimidine tracts are found on both sides of the alternative exon 3 of α-tropomyosin (TPM1), but, compared with the Src gene, they are longer and the pattern of regulation is different. Exons 2 and 3 of TPM1 are mutually exclusive (Figure 1). Exon 3 is used in most tissues because it contains strong splicing signals (Mullen et al, 1991), and the switch to exon 2 in smooth muscle cells is primarily the result of repression of exon 3 through the pyrimidine-rich tracts in the flanking introns (Gooding et al, 1994; Perez et al, 1997). It is likely that the longer pyrimidine tracts (P3 and DY) are bound by PTB in all cells (Singh et al, 1995; Perez et al, 1997; Gooding et al, 1998), but that the strong splicing signals override this except in smooth muscle cells. When the branch site is weakened by mutations, exon 3 is strongly repressed in HeLa cells (Gooding et al, 2006) by the P3 and DY PTB-binding elements (CG and CWJS, unpublished data). Figure 1.Sequences implicated in alternative splicing of exons 2 and 3 of TPM1. (A) Diagram of the alternative splicing patterns of exons 2 and 3. The lower pattern is a default pattern, and the upper is predominant in smooth muscle. Exons (boxes) and introns are not to scale; numbers refer to lengths in nucleotides. Splicing of exon 2 to exon 3 is prevented because the branch site (circle) in intron 2 is too close to exon 2. (B) Diagram of the transcripts used in this work. TM1 (562 nts) contains all the elements around exon 3 demonstrated to be involved in the regulation of splicing: P3, 50 nt polypyrimidine tract providing binding sites for splicing factor U2AF65 and regulatory protein PTB; URE, upstream regulatory element of repeated UGC motifs; DUGC, repeated UGC motifs on the 3′ side of the exon; DY, polypyrimidine tract of 40 nts, also bound by PTB. TM2 contains mutations converting the three putative high-affinity UCUU motifs in P3 to UUUU, UUUU and CCUU; TM3 contains two deletions totalling 12 nts in the DY tract; TM4 combines these mutations; TM1 trunc is truncated as shown. Download figure Download PowerPoint Although it is clear that PTB has a preference for binding to pyrimidine tracts, the connection between PTB binding and inhibition remains elusive. Two speculative models have dominated thinking, both of which have been invoked also to explain the activities of another repressor, hnRNP A1. One suggestion is that repressor molecules bound to sequences flanking the regulated sites associate in a complex, forcing the exon or site into a loop (Blanchette and Chabot, 1999; Chou et al, 2000; Eperon et al, 2000; Wagner and Garcia-Blanco, 2001; Nasim et al, 2002; Oberstrass et al, 2005; Spellman and Smith, 2006). However, direct evidence for this is lacking. Moreover, looping is not easily reconciled with evidence that some other exons are regulated by single-pyrimidine tracts (Shen et al, 2004; Izquierdo et al, 2005), and an alternative model is that extended complexes propagate along the RNA from the initial high-affinity site(s) (Eperon et al, 2000; Wagner and Garcia-Blanco, 2001; Zhu et al, 2001; Spellman and Smith, 2006). Given that estimates for the length of sequence bound or recognized by pure PTB vary so widely and that there is a strong concentration-dependent ability to form large complexes, it is impossible to infer the nature of the repressor complexes from the properties of the pure protein. A critical factor in developing mechanistic models for any regulated splicing reactions will be the ability to determine the numbers of proteins bound to RNA in complexes formed under splicing conditions. However, there are no methods available for doing this, whether with purified complexes or crude extracts, because of the complexity of the reactions: pre-splicing complexes assemble in the presence of numerous competing RNA-binding proteins, many of which have low specificity and are likely to bind dynamically and sometimes cooperatively. Such complexity may make it difficult to establish general principles for regulation, because different sites may involve different configurations of bound proteins. Despite considerable progress in identifying regulatory proteins and their target sites, we are in the unhappy situation that we do not know the molecular mechanisms operating at even one site. To address this critical limitation, we have developed single-molecule methods for analysing the stoichiometry of protein–RNA complexes formed in nuclear extracts. We show here that five or six molecules of PTB assemble around exon 3 of rat Tpm1 pre-mRNA, and we propose a model for the organization of the complex. This is the first report describing the measurement of the stoichiometry of proteins in complexes in nuclear extracts, and the method will be of widespread use in investigations of many aspects of gene expression. Results It has been shown previously that PTB binds to the pyrimidine-rich tracts flanking exon 3 and that these tracts are essential for repression (Gooding et al, 1994, 1998; Perez et al, 1997). To follow the binding of PTB to RNA among all the other proteins in nuclear extracts, GFP-labelled PTB (isoform 4) was expressed in HEK 293T cells and nuclear extracts were prepared. RNA transcripts corresponding to various portions of exon 3 and its flanking intron sequences (Figure 1) were transcribed from genomic fragments cloned in plasmid pGEM4Z (Gooding et al, 1998; Gromak et al, 2003a) cut with EcoRI or AccI (TM1 Trunc) and annealed to an oligoribonucleotide analogue complementary to the first nine nucleotides of the transcript that had been conjugated with a fluorescent label (Cy5) and biotin. Constructs TM2 and TM4 contain mutations in the three UCUU motifs in element P3 (Gromak et al, 2003a), and constructs TM3 and TM4 contained deletions of two short U-rich tracts in element DY (Gooding et al, 1998). The transcripts were incubated in the extracts under splicing conditions, and the complexes formed were captured from diluted reaction mixtures on streptavidin-treated silica slides. The signals from the GFP and Cy5 fluorophores were detected by total internal reflection fluorescence (TIRF) microscopy (Conibear and Bagshaw, 2000), separated with a beam splitter and acquired on two halves of an emCCD chip (Figure 2). Representative time courses of GFP fluorescence from single molecules of RNA are shown in Figure 3. The survival times of the fluorescence from single molecules were no shorter than those from GFP molecules deposited directly on the surface, indicating that fluorescence was terminated by bleaching rather than by dissociation from the complex. Figure 2.Representative image showing co-localization of RNA (red) and PTB protein (green). Nuclear extract containing GFP-PTB was incubated with TM1 RNA previously annealed to Cy5-labelled biotinylated oligonucleotide. The mixture was diluted and injected into a chamber on a prepared silica slide. Fluorescence from molecules attached to the surface was detected by TIRF. Images obtained in the Cy5 and GFP channels have been superimposed. White circles show co-localized Cy5 and GFP signals. Download figure Download PowerPoint Figure 3.Time recordings of GFP-PTB molecules. (A) Trace from GFP-PTB in nuclear extract in the absence of added RNA, showing the numbers of photons collected in frames of 300 ms versus time of observation. After about 23 s, the molecule bleached in a single step. (B) Trace from GFP-PTB co-localized with TM1 RNA. Five possible discrete steps are marked by lines. (C) As in (B), but in this case, discrete steps cannot be assigned. Download figure Download PowerPoint Quantification of PTB molecules in each transcript complex by single-step photobleaching Novel strategies were needed to quantify the numbers of GFP-PTB molecules associated with each transcript molecule. Unlike analyses done with membrane proteins (Leake et al, 2006; Ulbrich and Isacoff, 2007) or small organic fluorophores (Shu et al, 2007), counting the number of steps in which GFP molecules became progressively bleached was impracticable because the variations in amplitude between individual GFP molecules (see below) effectively masked the individual steps in many cases (compare Figures 3B and C). Selecting only those spots that produced clear steps would leave open the possibility of accidental bias. However, it was readily possible to distinguish complexes with only one GFP-PTB, which behaved like the individual GFP molecules seen in the absence of RNA (Figure 3A), from those containing more than one, which could be identified by the duration of the signal and the larger intensities (Figure 3C). This allowed us to count the numbers of complexes with one GFP-PTB and those with more than one. The total number of PTB molecules in the complexes could be calculated by assuming that (1) the total number of PTB molecules bound was constant and (2) the proportions of PTB and GFP-PTB followed a binomial distribution. The probability (p) that a molecule of PTB bound to RNA was GFP-PTB, rather than endogenous unlabelled PTB, was determined by cross-linking of protein in the extract to radioactive TM1 trunc RNA (Figure 4) and found to be about 0.45. Figure 4.Cross-linking of PTB and GFP-PTB in nuclear extracts. SDS–PAGE analysis of proteins labelled with 32P by cross-linking to radioactive RNA. The nuclear extract from 293T cells transfected with plasmid expressing GFP-PTB was the same as that used throughout this work. The other nuclear extracts were from untransfected HeLa and 293T cells, as shown. The TPM1 substrate was TM1 trunc; in lanes labelled C, the extracts were incubated with an adenovirus pre-mRNA used as a substrate for splicing in vitro. Download figure Download PowerPoint The results from the analysis of about 1600 molecules (Table Ia) show that the fraction of RNA molecules associated with GFP-PTB bleaching in a single step rose from 18%, with the longest wild-type transcript, to 68% with the shortest. Table Ib shows the fraction of molecules expected to show single-step bleaching with p=0.45. The observed value of 18% for the wild-type transcript TM1 falls between the values expected for 5 or 6 molecules of PTB. Similarly, the complexes formed on TM2 and TM3 contain 4–5 molecules, those on TM4 contain 3–4 and those on TM1 trunc contain 2. The observation that removing the three high-affinity UCUU motifs from the P3 element (TM2) reduced the number of bound PTB molecules by only one was surprising and suggested that the common practice of predicting PTB-binding sites by high-affinity motifs is misleading. Table 1. Observed proportions of Cy5 (RNA) molecules in which associated GFP-PTB is bleached in a single step Substrate TM1 TM2 TM3 TM4 TM1 trunc % Single step 18 (26%) 28 (36%) 29 (39%) 40 (47%) 68 (71%) No. measured 472 288 319 342 159 The fraction of GFP spots that co-localized with Cy5 spots and exhibited single step bleaching is shown for each substrate tested (% single step). The values in parentheses show the highest value for the fraction when ambiguous assignments are included (see Materials and methods). The total number of measurements with each RNA substrate is shown. Table 2. Proportions of molecules showing single step bleaching predicted with various values of n Total number of PTB/RNA (b) 6 5 4 3 2 1 p=0.45 14 22 33 49 71 100 Theoretical values for the fraction of spots exhibiting a single bleaching behaviour were calculated using a binomial distribution for the complexes formed by b bound PTB molecules with the fraction of PTB-GFP molecules equal to 0.45. The fraction of GFP spots exhibiting single bleaching in extract in the absence of any RNA substrate was >90%. Table II shows the fraction of labelled RNA molecules co-localized with GFP-PTB (Table II), which reflects the affinity of the interactions. The fractions were reduced progressively by mutations in either polypyrimidine tract (TM2 and TM3), both tracts (TM4) and by truncation of the RNA immediately downstream of P3 (TM1 trunc). These results provide an independent confirmation of the significance of the single-step photobleaching results in Table Ia. Table 3. Efficiency of formation of the complexes for various substrates TM1 TM2 TM3 TM4 TM1 trunc Oligonucleotide No. Cy5 spots 334 657 866 1344 824 1724 % Associated with GFP-PTB 66 47 52 32 23 15 nts from any possible site for RRMs 1–3, RRM4 binding does not provide a constraint and was not taken into account. Analysis of the entire sequence of TM1 showed that there were numerous overlapping candidate sites, almost entirely located within the known P3 and DY-binding elements (Figure 6A). The maximum number of proteins that could be accommodated simultaneously in P3 was three, although there are a number of possible arrangements (Figure 6B). To test whether up to three proteins could bind in practice, gel shift assays were done with TM1 trunc RNA and various proportions of recombinant N-terminally truncated PTB1 and full-length PTB4 proteins at a saturating total PTB concentration of 4 μM, in the absence of nuclear extract. The results confirm that this region could accommodate three PTB molecules (Figure 6C). Figure 6.Ways of accommodating five PTB molecules on TM1 pre-mRNA. (A) Possible binding sites for PTB monomers (domains 1–3), based on the sequence specificities inferred from the structures (Oberstrass et al, 2005). Each vertical line represents the first nucleotide in the potential binding site. The ordinate shows the number of potential binding sites that begin at the same nucleotide; such sites arise because of the range of values allowed for the distance between the motifs recognized by each domain. The abscissa shows the position in the regulatory region, with the P3 and DY elements and exon 3 delineated. (B) Possible arrangements for the binding of three molecules of PTB to the P3 element. Each horizontal line shows one possible arrangement. Coloured bars show the region bound by the three separate molecules in each arrangement. Identical arrangements arise on different horizontal lines when RRM2 is predicted to bind at different positions within a monomer but RRM domains 1 and 3 are at the same positions. The region marked ends at the last specific nucleotide recognized by domain 3 and does not include the two additional nucleotides bound by the domain (Oberstrass et al, 2005). (C) Gel shift analysis of PTB binding capacity of TM1 trunc RNA. The left-hand lane contained RNA only. The other lanes contained, from the left, RNA incubated with His-tagged PTB4 at 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 and 0 μM, and sufficient His-tagged PTB1-N (containing a deletion of the N-terminal 54 amino acids) to maintain a total PTB concentration of 4 μM. Electrophoresis was done on a 4% polyacrylamide gel. Labels to the right of the image show our interpretation of the compositions of the bands. (D) Gel shift analysis of PTB binding capacity of mutant TM1 trunc RNA. The substrate is derived from TM2 and contains the mutations in the P3 element that change the three putative high-affinity UCUU motifs in P3 to UUUU, UUUU and CCUU and strongly reduce repression. The protein concentrations and conditions of electrophoresis are as in Figure 6C. (E) Possible arrangements for the binding of two PTB molecules to the DY element, labelled as in (B), in which both molecules are located at optimal sites. (F) Gel shift analysis of PTB binding capacity of RNA from the DY element. The transcript contains nucleotides 449–562, that is, all of the DY element shown in Figure 6E. The protein concentrations and conditions of electrophoresis are as in Figure 6C. Possible assignments are shown. Download figure Download PowerPoint Table 4. Values of n, the number of PTB molecules per molecule of RNA, inferred from the best fits to cumulative distributions of total photon counts Substrate TM1 TM2 TM3 TM4 TM1 trunc n 5–6 4–5 4–5 3–4 2 Results from Tables Ia and Ib and Figures 5C–G. The correspondence between the binding we observed in nuclear extract and the structure-based predictions was underscored by the mutant TM2, in which mutations had altered all three of the UCUU sequence motifs previously believed to be the high-affinity binding sites in P3 (Perez et al, 1997). These mutations reduce repression of exon 3 in smooth muscle (Gromak et al, 2003b) and HeL
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