Ser/Arg-rich Protein-mediated Communication between U1 and U2 Small Nuclear Ribonucleoprotein Particles

Boukis, Lisa A.; Liu, Nan; Furuyama, Suzanne; Bruzik, James P.

doi:10.1074/jbc.m313209200

Cited by 25 publications

(19 citation statements)

References 57 publications

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“…Recent studies have shown that the RS domain of SR proteins bound to the exon can also interact directly with the BPS (6). Finally, SR proteins not only function by binding to ESEs, but they also play a critical role in the basic splicing mechanism within the intron, either by facilitating interactions between proteins bound to the 5Ј and 3Ј splice sites within the intron (24)(25)(26) and͞or by contacting the 5Ј splice site in the mature spliceosome (27).…”

Section: Discussionmentioning

confidence: 99%

Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers

Ibrahim

Schaal

Hertel

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

112

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The 5 and 3 splice sites within an intron can, in principle, be joined to those within any other intron during pre-mRNA splicing. However, exons are joined in a strict 5 to 3 linear order in constitutively spliced pre-mRNAs. Thus, specific mechanisms must exist to prevent the random joining of exons. Here we report that insertion of exon sequences into an intron can inhibit splicing to the downstream 3 splice site and that this inhibition is independent of intron size. The exon sequences required for splicing inhibition were found to be exonic enhancer elements, and their inhibitory activity requires the binding of serine͞arginine-rich splicing factors. We conclude that exonic enhancers can act as barriers to prevent exon skipping and thereby may play a key role in ensuring the correct 5 to 3 linear order of exons in spliced mRNA.intronic splicing silencers ͉ pre-mRNA splicing ͉ accurate splice-site recognition and pairing M ost genes in higher eukaryotes contain multiple introns and exons that are transcribed to generate pre-mRNA. The production of mature mRNA requires the precise removal of introns and joining of exons by splicing. In constitutively spliced pre-mRNAs all of the introns are excised, and the exons are joined in the correct 5Ј to 3Ј order. This is a consequence of the fact that 5Ј splice sites are ligated to a 3Ј splice site exclusively within the same intron, thus avoiding the exclusion of exons (''exon skipping''). Two key problems in understanding the mechanisms of accurate pre-mRNA splicing are how the splicing machinery recognizes exons and how exons are joined in the correct 5Ј to 3Ј order (i.e., how exon skipping is avoided).Significant advances have been made in understanding the mechanisms involved in accurate exon recognition (1-3). This process requires weakly conserved sequence elements located at the 5Ј and 3Ј splice sites and at the branchpoint sequence (BPS) within introns. In addition, exons contain sequence elements known as exonic splicing enhancers (ESEs), which function as binding sites for members of the serine͞arginine-rich (SR) family of splicing factors. The bound SR proteins promote the use of flanking 5Ј and 3Ј splice sites through both protein-protein and protein-RNA interactions (4-6). Although splicing enhancer͞SR protein complexes provide a mechanism for exon recognition, the mechanisms by which exon skipping is avoided are not understood.An important insight into this problem was provided by the observation that many human genetic diseases are the consequence of single base mutations in ESEs and that these mutations cause exon skipping (7). Further insights were provided by studies of regulated alternative splicing, where it was shown that the inclusion of an alternate exon requires an ESE and the presence of specific SR proteins (8). Thus, exonic enhancer͞SR protein complexes are required to avoid exon skipping in both constitutive and alternative splicing. Although these observations indicate that exon recognition is a necessary step in avoiding exon skipping, it ...

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Section: Discussionmentioning

confidence: 99%

Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers

Ibrahim

Schaal

Hertel

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

112

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“…During exon definition, splice sites are recognized across the exon through the binding of U1 snRNP to the 59 splice site and U2AF and U2 snRNP binding to the upstream 39 splice site (Hoffman and Grabowski 1992;Chiara and Reed 1995;Boukis et al 2004;Sharma et al 2008). Splicing enhancers and silencers modulate the stability of these complexes, presumably by promoting or interfering with RS domain contacts (Graveley et al 2001;Boukis et al 2004). Exon defined splice sites must then transition to pair across the intron to mediate the assembly of the catalytic spliceosome.…”

Section: Components Of the Exon Definition Complex Are Retained On Spmentioning

confidence: 99%

Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns

Crabb¹,

Lam²,

Hertel³

2010

RNA

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The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites.

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“…Associations between the U1 snRNP protein U1 70k and the 39 splice site-bound protein U2AF have also been reported. This interaction was proposed to be mediated by the SR protein SC35 in mammals but by a direct contact in yeast (Wu and Maniatis 1993;Abovich and Rosbash 1997;Reed 2000;Boukis et al 2004). In HeLa cell extracts, the SR-related proteins Srm160 and Srm300 have also been shown to bridge the U1 and U2 snRNPs (Eldridge et al 1999;Blencowe et al 2000).…”

mentioning

confidence: 99%

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Sharma

Wongpalee

Vashisht³

et al. 2014

Genes Dev.

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The pairing of 59 and 39 splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 59 splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 39 splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 59 and 39 splice site complexes within the assembling spliceosome.[Keywords: RNA-protein interaction; alternative splicing; gene expression; pre-mRNA splicing; ribonucleoprotein; snRNA; spliceosome] Supplemental material is available for this article. Intron removal is catalyzed by an ;40S RNP complex called the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4, U5, and U6) and ;150 auxiliary proteins . In vitro, the spliceosome assembles onto an intron through the sequential binding of the snRNPs Hoskins et al. 2011). The 59 splice site is recognized by the U1 snRNP through base-pairing between the pre-mRNA and the snRNA. At the 39 end of the intron, the U2 auxiliary factor (U2AF) and splicing factor 1 (SF1) bind to the 39 splice site and branch point sequence, respectively. Initial association of the U2 snRNP can be ATP-independent and has been proposed to occur through interactions with the U2AF65 protein (Gozani et al. 1998;Das et al. 2000). This ATP-independent complex is referred to as the E complex. However, the stable association of U2 with the pre-mRNA requires ATP hydrolysis and formation of a base-pairing interaction between U2 snRNA and the branch point sequence with displacement of SF1. This pre-mRNP complex containing both the U1 and U2 snRNPs is called the prespliceosomal A complex. Recruitment of the U4/U6-U5 tri-snRNP forms the precatalytic B complex. In another mode of B complex formation, the U4/U6-U5 trisnRNP binds with the U2 snRNP to the 39 splice site, Ó 2014 Sharma et al. This article is distributed exclusively by Cold Spring...

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Ser/Arg-rich Protein-mediated Communication between U1 and U2 Small Nuclear Ribonucleoprotein Particles

Cited by 25 publications

References 57 publications

Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers

Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers

Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

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