Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

Self Cite

305

254

SV40 early pre-mRNA is alternatively spliced to produce large T and small t mRNAs by use of different 5'-splice sites and a shared 3'-splice site. The large T splicing pathway uses multiple lariat branch sites, whereas small t splicing, constrained by its small intron size, can use only one. We exploited this situation to test the hypothesis that RNA-RNA base pairing between U2 snRNA and the branch site sequence is important in mammalian pre-mRNA splicing by constructing and analyzing several mutations in the small t pre-mRNA branch site (UUCUAAU). All of the mutations resulted in substantial decreases in small t splicing relative to large T. To test whether these effects resulted from decreased base pairing with U2 snRNA, compensatory mutations were introduced at the appropriate positions (nucleotides 34-36) in a cloned human U2 gene. All branch site mutations tested (four separate single base substitutions and two triple mutations) were suppressed (i.e., small t splicing was increased) by the appropriate U2 mutations. These results establish that recognition of the poorly conserved mammalian pre-mRNA branch site sequence by U2 snRNP can involve base-pairing.[Key Words: snRNP; base pairing; small t mRNA; large T mRNA]

supporting

confidence: 57%

Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing.

Wu¹,

Manley²

1989

Self Cite

305

254

“…The choice between these mutually exclusive sites is dependent on cell-specific, trans-acting factors. Additional sites required in cis for splice site selection have been implicated in studies on other loci, including the branch site (Noble et al 1987;Fu et al 1988) and sequences within the alternatively spliced exons (Somasekhar and Mertz;Mardon et al 1987;Barone et al 1989;Hampson et al 1989;Laski and Rubin 1989;Streuli and Saito 1989). This diversity of regions involved in regulating splice choice suggests that multiple mechanisms exist for controlling RNA processing.…”

mentioning

confidence: 99%

Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns.

Nagoshi¹,

Baker²

1990

158

119

Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wildtype sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.

“…Therefore, regulated alternative pre-mRNA processing must involve either differences in the constitutive cis-and trans-acting splicing elements or additional specific elements. Among the cis elements shown to influence alternative splice site selection are the relative homology to the consensus sequences for the 5' splice site (Eperon et al 1986~ Zhuang et al 1987) and the branchpoint {Reed and Maniatis 1988;, the relative proximity of these elements (Fu et al 1988a;Smith and Nadal-Ginard 1989}, the nature of the polypyrimidine tract {Fu et al 1988b}, the sequences between the 3' splice site and the branchpoint/polypyrimidine tract {Emeson et al 1989;Goux-Pelletan et al 1990;Helfman et al 1990}, the exon sequences (Reed and Maniatis 1986~ Mardon et al 1987~ Streuli and Saito 1989}, and the secondary structure {Solnick 1985; Libri et al 1990). Much less is known about the trans factors that regulate alternative splicing.…”

mentioning

confidence: 99%

Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice.

Mullen

Smith²,

Patton³

et al. 1991

148

132

We have used exons 2 and 3 of the rat a-tropomyosin gene to analyze the basis of mutually exclusive exon selection. The basis of the strict mutually exclusive behavior of this exon pair is enforced by the proximity of the exon 3 branchpoint to the 5' splice site of exon 2. With the exception of smooth muscle cells, exon 3 rather than exon 2 is incorporated into mRNA in all cell types. We show here, using both in vivo and in vitro cell-free systems, that this alternative exon selection is a consequence of general principles that govern 3' splice site selection. In the absence of exon 3, exon 2 is utilized efficiently in all cells. Selection of exon 3 is therefore the default result of a competition between exons 2 and 3 for the flanking constitutive splice sites. The basis of this competition is the relative strength of the polypyrimidine tract/branchpoint elements of the two exons. The major determinant of this splice site strength is the pyrimidine content adjacent to the branchpoint, and this involves no other sequence specificity. The branchpoint elements play an important but secondary role. The functional strengths of the different polypyrimidine tract/branchpoint combinations, as determined in cis competition assays, showed a perfect correlation with their binding affinities to a spliceosome component that interacts with the pre-mRNA in an ATP-independent manner. Selection of exon 3 in most cell types therefore reflects the preferential interaction of these splice site elements with constitutive splicing factors early in spliceosome assembly. The aspects of splice site selection analyzed here are likely to be of general applicability to constitutive and alternative pre-mRNA splicing.[Key Words: Rat c~-tropomyosin gene; exon selection; branchpoint polypyrimidine tracts; splice site selection] Received December 10, 1990; revised version accepted February 1, 1991.One of the major unresolved issues conceming premRNA splicing is the problem of splice site selection. Most eukaryotic genes possess multiple intervening sequences, or introns, each bounded by a pair of functional 5' and 3' splice sites. The open reading frame of the mature mRNA is preserved by the accurate excision of these introns from the primary transcript. Potentially, splice sites could pair in many different combinations disruptive to message integrity. That this does not occur to any great extent is testimony to the high fidelity of the splicing process. The role of limited consensus sequences at the ends of introns in specifying splice sites has long been recognized [Mount 1982). These elements include the practically invariant GU and AG dinucleotides at the 5' and 3' splice sites, respectively, the 5' splice site consensus sequence that base-pairs with U1 snRNA [Zhuang and Weiner 1986), the branchpoint consensus sequence that base-pairs with U2 snRNA {Parker et al. 1987; Wu and Manley 1989;, and the polypyrimidine tract between the branchpoint and the 3' splice site AG. However, comparisons of primary sequences have yet to produce a set of ...