Defining a 5??? splice site by functional selection in the presence and absence of U1 snRNA 5??? end

Lund, Mette K.; Kjems, Jørgen

doi:10.1017/s1355838202010786

Cited by 80 publications

(82 citation statements)

References 55 publications

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“…The 5Ј end of U1 snRNA is complementary to the consensus sequence of the 5Ј splice site, and, indeed, U1 snRNA base pairs with the 5Јss as a prerequisite step for splicing (Lerner et al 1980;Rogers and Wall 1980;Zhuang and Weiner 1986;Wassarman and Steitz 1992;Alvarez and Wise 2001). This model still holds, in view of other studies suggesting that the base-pairing of U1 snRNA with the 5Јss is nonessential for splicing (Bruzik and Steitz 1990;Crispino et al 1994;Hwang and Cohen 1996;Du andRosbash 2001, 2002;Lund and Kjems 2002), which are likely to be the exception (Maroney et al 2000).…”

Section: Introductionmentioning

confidence: 87%

“…The idea of a minimal number of base pairs was also derived from another study (Zhuang and Weiner 1986). Additionally, when the number of U1 snRNA:5Јss base pairs is relatively high (>7), the reaction efficiency is reduced under low RNA concentrations in humans (Lund and Kjems 2002). In S. cerevisiae, hyperstabilization of the U1 snRNA:5Јss base-pairing with 10 bp was detrimental (Staley and Guthrie 1999).…”

Section: Introductionmentioning

confidence: 99%

“…This also suggests that the different positions of the 5Јss might have a mutual relationship: A mispair between U1 snRNA and one position of the 5Јss is compensated by a base pair of U1 snRNA to another position(s), thus maintaining the base pairs' number above the minimum. Indeed, evidence for such a mutual relationship among the 5Јss positions were found: The existence of G at position +3 depends on the concordance that the residues at positions +4 to +6 have with U1 snRNA (Ohno et al 1999); base-pairing of U1 snRNA with positions +3 and +4 compensates for a mispair of U1 snRNA with position +5 (Nelson and Green 1990); and the dependence of positions +5 with position +3 (Burge and Karlin 1997;Clark and Thanaraj 2002) and with positions +3 and +4 was demonstrated (Lund and Kjems 2002). Additionally, a linkage between the exonic and intronic portions of the 5Јss was indicated in a few studies (Burge and Karlin 1997;Rogozin and Milanesi 1997;Thanaraj and Robinson 2000).…”

Section: Introductionmentioning

confidence: 99%

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Comparative analysis detects dependencies among the 5′ splice-site positions

Carmel¹,

Tal²,

Vig³

et al. 2004

RNA

197

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Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5 splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5 splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, −2 and +5, and −1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5 splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5 splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5 splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5 splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and −1 is the only functional requirement for mRNA splicing of this 5 splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5 splice site.

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Section: Introductionmentioning

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comparative analysis detects dependencies among the 5′ splice-site positions

Carmel¹,

Tal²,

Vig³

et al. 2004

RNA

197

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“…Although these mutant complexes are much less stable than WT complexes, they still maintain considerable sequence specificity for a proper 5Јss. The canonical RNA-RNA base-pairing is also not essential for either 5Јss selection or splicing in the HeLa in vitro system (21). All of these data indicate that other factors, probably U1 snRNP proteins, make significant contributions to 5Јss recognition and complex formation in mammals and yeast.…”

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confidence: 90%

Effects of the U1C L13 mutation and temperature regulation of yeast commitment complex formation

Tardiff

Moore

et al. 2004

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

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The U1 small nuclear ribonucleoprotein particle U1C protein has a zinc finger-like structure (C2H2 motif) at its N terminus, which is conserved from yeast to humans. Mutations of amino acid L13 within this domain rescue the essential function of the helicase protein Prp28p. Prp28p has been implicated in unwinding the 5 splice site (5ss)-U1 small nuclear RNA (snRNA) base-pairing, to allow replacement of U1 snRNA with U6 snRNA during spliceosome assembly. The L13 phenotype has therefore been interpreted to indicate that WT U1C contributes to 5ss-U1 snRNA stabilization by binding to the RNA duplex. We show here that an L13 mutant extract cannot form stable base-pairing at room temperature but is permissive for U1-5ss base-pairing at low temperature. This phenotype is similar to that of a U1C-depleted extract, indicating that the U1C L13 mutation is a strong loss-of-function mutation. The two mutant extracts are unlike a WT extract, which undergoes stable pairing at room temperature but little or no pairing at low temperature. Taken together with previous results and the failure to observe a direct interaction of U1C with the U1-5ss duplex, the data suggest that U1C contributes indirectly to stable U1-5ss base-pairing under permissive conditions. A model is proposed to account for the L13 results. E ukaryotic genes are usually interrupted by introns, which must be precisely excised. Intron removal or pre-mRNA splicing takes place within a large RNA-protein machine termed the spliceosome. The five splicing small nuclear ribonucleoprotein particles (snRNPs) and many additional non-snRNP spliceosomal proteins play specific roles in the splicing process (1-8). In both yeast and mammals, pre-mRNA recognition by the spliceosome relies on a set of consensus sequences within the pre-mRNA intronic regions. These are the 5Ј splice site (5Јss), branchpoint, and 3Ј splice site regions, which associate with protein and RNA components of the spliceosome (2, 3, 9, 10).During in vitro splicing, U1 snRNP recognizes the 5Јss in an ATP-independent fashion and joins the pre-mRNA to form commitment complex in yeast or the E complex in mammals (11,12). Because spliceosome assembly is cotranscriptional and the 5Јss is synthesized before the other two cis-acting regions, it presumably associates with U1 snRNP before other splicing components join during subsequent steps of spliceosome assembly (13,14). An alternative view is that a preassembled pentasnRNP recognizes the 5Јss, and the subsequent steps of ''spliceosome assembly'' occur as conformational changes in vivo (15). In either case, the 5Јss sequence functions not only in initial intron recognition but also in subsequent splicing steps, such as splice site partner assignment and even catalysis (16).Commitment complex and E complex formation involve basepairing between the highly conserved pre-mRNA 5Јss and the single-stranded 5Ј end of U1 small nuclear RNA (snRNA). This base-pairing is critical for the U1 snRNP-5Јss interaction and contributes significantly to 5Јss selection, in both...

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“…U1 snRNP binds to the 5Ј-splice site by a combination of RNA/RNA and protein/RNA interactions (49,50). The 3Ј-splice site is initially recognized by U2AF.…”

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confidence: 99%

Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing

Lam

Bakshi

Ekinci

et al. 2003

Journal of Biological Chemistry

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The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5 -splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3 -splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3 -splice site choice. However, little is known about enhancer-dependent regulation of alternative 5 -splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5 -splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated femalespecific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5 -splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5 -and 3 -splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5 -and 3 -splice sites.Alternative pre-mRNA splicing is commonly used to regulate the expression of genes and to enrich the proteomic diversity of higher eukaryotic organisms (1-4). Current estimates suggest that ϳ60% of human genes are alternatively spliced (5), sometimes leading to thousands of different mRNA isoforms from a single gene. Numerous examples describe how alternative splicing regulates gene expression in humans, but the mechanisms involved are understood in only a few cases (6 -17). In contrast, the use of genetics in Drosophila has aided in the identification of proteins that regulate alternative splicing. In particular, the genes involved in Drosophila sex determination have become prototypes for the study of positive and negative control of alternative splicing (18). The best characterized gene is the doublesex (dsx) 1 gene, in which the use of two alternative 3Ј-splice sites leads to the expression of male-or female-specific gene products that initiate gender-specific sexual differentiation. Female-specific splicing of dsx requires the production of two proteins, Transformer (TRA) and Transformer2 (TRA2), and an exonic splicing enhancer (ESE) element located within the untranslated region of the fourth exon (19 -21). Both TRA and TRA2 contain Arg/Ser-rich (RS) domains, protein interaction domains characteristic of the Ser/Arg-rich (SR) family of essential splicing factors. SR prot...

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Defining a 5??? splice site by functional selection in the presence and absence of U1 snRNA 5??? end

Cited by 80 publications

References 55 publications

Comparative analysis detects dependencies among the 5′ splice-site positions

Comparative analysis detects dependencies among the 5′ splice-site positions

Effects of the U1C L13 mutation and temperature regulation of yeast commitment complex formation

Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing

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