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An Exonic Splicing Silencer Represses Spliceosome Assembly After ATP-Dependent Exon Recognition

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An Exonic Splicing Silencer Represses Spliceosome Assembly After ATP-Dependent Exon Recognition ARTICLES An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition Amy E House & Kristen W Lynch Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome’s dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 http://www.nature.com/nsmb does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4–U6–U5 tri-snRNP–containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway. Virtually every mammalian precursor (pre)-mRNA transcript is exon (that is, exon recognition) to the cross-intron interaction (that is, spliced, through the removal of intronic sequences and the ligation exon pairing) that eventually is required for catalysis3. In particular, E, of exonic sequences, to generate a mature protein-coding mRNA A and B complexes have primarily been analyzed and defined in transcript. This splicing reaction is catalyzed by a complex, macro- studies using simple two-exon/single-intron constructs, such that the molecular machine known as the spliceosome that is comprised of five spliceosomal components can only assemble across the intron. How- Nature Publishing Group Group Nature Publishing 1 6 snRNAs and several hundred associated proteins . Each snRNA (U1, ever, a number of studies have shown that at least the U1 and U2AF U2, U4, U5 and U6) forms the core of a specific snRNP component subunits of the spliceosomal E complex can interact across a single 200 © and promotes formation of the spliceosome through base-pairing exon as part of an exon-definition complex that is essential for high- interactions with splice site sequences in pre-mRNA templates or with fidelity splice site recognition4,5. Likewise, the U1- and U2-containing other snRNAs, or both2. Importantly, the mature, catalytic spliceo- A complex has been defined only as a cross-intron complex, a view some (complex C) is not a preformed enzyme; rather, it assembles on supported by at least one study that has demonstrated stable pairing of the pre-mRNA in a stepwise and highly dynamic manner. In vitro exons concurrent with A complex formation6. Nevertheless, in experi- studies have identified several distinct intermediates along this assem- ments in which two exons are artificially separated from one another, a bly pathway (E-A-B)2,3. The earliest complex committed to the U1- and U2-containing complex can form on an isolated exon and splicing pathway (E) has been defined as U1 snRNP bound to a then pair with an exogenous exon to result in a catalytically active 5¢ splice site with the U2 snRNP auxiliary factor (U2AF) bound to the spliceosome7. Therefore, it is possible for the U1 and U2 snRNPs to 3¢ splice site. E complex is then chased into A complex in the first ATP- first interact across a single exon before pairing with spliceosomal dependent step in assembly, which involves the binding of U2 snRNP components on flanking exons, indicating that a U1- and U2-contain- to the branchpoint sequence at the 3¢ splice site. Subsequently, ing (that is, A-like) exon-definition complex may be able to form on B complex is formed by the recruitment of the U4–U6–U5 natural substrates as part of the spliceosome assembly pathway2,3. tri-snRNP complex. C complex finally forms by extensive remodeling Although spliceosome assembly and catalysis occurs with high of both the RNA and protein components of B complex to generate the fidelity, it is also subject to intricate regulation. Indeed, regulated active enzymatic spliceosome capable of catalyzing the splicing reaction. inclusion or exclusion of exons, by the process of alternative splicing, Isolation of the distinct E, A and B intermediate complexes along has been predicted to be a primary mechanism for regulating gene the spliceosome assembly pathway has allowed for the study of specific expression2. In particular, exonic splicing enhancers (ESEs) and steps in the splicing process, such as exon recognition, selection of splicing silencers (ESSs) respectively promote and inhibit spliceosome splice sites and catalysis2,3. However, such studies do not preclude the recognition of a regulated exon through the activity of associated existence of other spliceosome-assembly intermediates and do not regulatory proteins. Previous studies of the mechanism of exon provide details as to crucial transitions in spliceosome assembly, such repression by the ESS class of sequences have shown that regulation as the transition from spliceosomal subunits interacting across an typically occurs during the initial ATP-independent recognition of Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, USA. Correspondence should be addressed to K.W.L. ([email protected]). Received 13 March; accepted 30 August; published online 24 September 2006; doi:10.1038/nsmb1149 NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 10 OCTOBER 2006 937 ARTICLES Figure 1 ESS1 represses splicing of CD45 exon 4 a c alt-ESS∆E3 in vitro by stalling spliceosome assembly at an WT mESS1 alt-ESS –––+ + + : ESS1 comp unusual step. (a) RNA derived from CD45 mESS1∆E3 minigenes containing regulated exon 4 (with alt∆E3 m∆E3 : ESS1 comp WT, mESS or alt-ESS sequence) flanked by –+ –+ constitutive exons 3 and 7 was incubated in JSL1 nuclear extract in the absence (–) or 43 39 36 30 : % spliced presence (+) of exogenous competitor (comp) 227 21 22 2 2 : % 3-exon ESS1 RNA for 110 min, and products were ∆ analyzed by low-cycle RT-PCR16.Accuracyofall bdWT E3 WT∆E3 spliced products was confirmed by cloning and alt-ESS∆E3 –ESS1 +ESS1 : Comp RNA sequencing. % 3 exon, percentage of three-exon 0 5 15 30 60 90 120 0 5 15 30 60 90120 : Time (min) mESS1∆E3 products. (b)Top,RNAderivedfromtheWTDE3 WT∆E3 alt∆E3 m∆E3 minigene was spliced as in a. For quantification, –––+ : ESS1 comp 01030010 30 0 30 0 30 : Time (min) see Figure 6a.Bottom,32P-labeled WTDE3 RNA B was incubated in JSL1 nuclear extract under B A splicing conditions in the absence (–) or presence * * A (+) of exogenous ESS1 competitor RNA, and H/E H/E resulting spliceosome assembly complexes were resolved on a nondenaturing polyacrylamide gel. Comigrating H and E complexes (H/E), A and B complexes and the stalled complex (asterisk) are labeled. In b, B complex in the presence of ESS is more diffuse at 15–60 min than in d, because we held these reactions on ice with heparin for the remainder of the extended time course before loading. (c) Splicing of RNA derived from alt-ESSDE3 (altDE3) and mESS1DE3 (mDE3) minigenes. Splicing reactions for these and all subsequent experiments were terminated at 110 minutes. (d) Spliceosome assembly gels as in b,using32P-labeled versions of the RNAs shown. http://www.nature.com/nsmb splice sites by U2AF or the U1 snRNP3,8–11. However, the inherently skipping of the CD45 variable exon 4 both in vivo and in vitro15,16. dynamic nature of the spliceosome suggests the potential for biologi- In vitro splicing reactions using a minigene containing the wild-type cally relevant regulation of splicing at any of the intermediates along exon 4 sequence flanked by the CD45 constitutive exons 3 and 7 the spliceosome assembly pathway. (Fig. 1a, WT) show a low level of exon 4 inclusion, or ‘three-exon’ The CD45 gene is an excellent model system for understanding the product. However, upon addition of exogenous ESS1 RNA, there is a mechanism and regulation of alternative splicing events. CD45 is a marked increase in the level of exon inclusion (Fig. 1a, +), consistent transmembrane protein tyrosine phosphatase whose activity is crucial with the exogenous ESS1 titrating functionally important ESS1-bind- for T-cell development and signaling12. The gene encoding CD45, ing proteins away from the pre-mRNA substrate, thereby alleviating 16 Nature Publishing Group Group Nature Publishing PTPRC (hereafter called CD45)containsthreevariableexons(exons4, repression. As expected from previous studies , the ability of exo- 6 5 and 6) that are alternatively spliced in human T cells, resulting in genous ESS1 to promote exon inclusion is dependent on the regulated 13 200 distinct isoforms that differ in activity .Previousanalysisofcis-acting exon containing a functional ESS1 element. Splicing of the mESS1 © elements that govern the regulation of exon 4 splicing has identified an minigene, which contains point mutations in ESS1 that abolish exonic silencer element (ESS1) that is required for exon repression14,15. silencing activity15, results in an increased basal level of exon inclusion Moreover, at least three heterogeneous nuclear RNPs (hnRNPs) bind that cannot be further enhanced by addition of competitor ESS1 RNA specifically to a core sequence within ESS1, and at least one of these, (Fig. 1a, mESS1). Similarly, the addition of ESS1 competitor has no hnRNP L, functions to repress exon splicing16. However, as the effect on the splicing of a minigene in which the entire ESS1 element is mechanism by which hnRNP L functions to block inclusion of removed and replaced with a heterologous sequence from CD45 exon exon 4 has been unknown, we set out to identify the stage of 14 that binds hnRNP A1 and A2 (UTSWMC; A.
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