Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Ast, Gil; Pavelitz, Thomas; Weiner, Alan M.

doi:10.1093/nar/29.8.1741

Cited by 15 publications

(25 citation statements)

References 89 publications

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“…What may underlie this signal? Previous studies have indicated that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with an anchoring site, located upstream of the BS (Gozani et al 1996;Kramer 1996;Ast et al 2001). Thus, this signal may serve as such an anchoring site.…”

Section: Splicing In Y Lipolyticamentioning

confidence: 99%

Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes

Schwartz

Silva²,

Burstein³

et al. 2007

Genome Res.

166

174

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Introns are among the hallmarks of eukaryotic genes. Splicing of introns is directed by three main splicing signals: the 5Ј splice site (5Јss), the branch site (BS), and the polypyrimdine tract/3Јsplice site (PPT-3Јss). To study the evolution of these splicing signals, we have conducted a systematic comparative analysis of these signals in over 1.2 million introns from 22 eukaryotes. Our analyses suggest that all these signals have dramatically evolved: The PPT is weak among most fungi, intermediate in plants and protozoans, and strongest in metazoans. Within metazoans it shows a gradual strengthening from Caenorhabditis elegans to human. The 5Јss and the BS were found to be degenerate among most organisms, but highly conserved among some fungi. A maximum parsimony-based algorithm for reconstructing ancestral position-specific scoring matrices suggested that the ancestral 5Јss and BS were degenerate, as in metazoans. To shed light on the evolutionary variation in splicing signals, we have analyzed the evolutionary changes in the factors that bind these signals. Our analysis reveals coevolution of splicing signals and their corresponding splicing factors: The strength of the PPT is correlated to changes in key residues in its corresponding splicing factor U2AF2; limited correlation was found between changes in the 5Јss and U1 snRNA that binds it; but not between the BS and U2 snRNA. Thus, although the basic ability of eukaryotes to splice introns has remained conserved throughout evolution, the splicing signals and their corresponding splicing factors have considerably evolved, uniquely shaping the splicing mechanisms of different organisms.[Supplemental material is available online at www.genome.org.]Splicing of pre-mRNA is a key step in eukaryotic gene expression, contributing to gene regulation, protein diversity, and phenotypic complexity. Introns are removed from the pre-mRNA by the spliceosome, which is composed of five snRNPs (small nuclear ribonucleoprotein) (U1, U2, U4, U5, and U6), each containing a small RNA bound by proteins. High-precision recognition of introns is required for correct splicing. This recognition is achieved by the binding of splicing factors to signals of varying specificity that are located both in the intron and its flanking exons (Hastings and Krainer 2001;Black 2003;. In vertebrates, three signals are known to direct splicing: The 5Ј splice site (5Јss) at the 5Ј end of the intron, the polypyrimdine tract/3Ј splice site (PPT-3Јss) at the 3Ј end of the intron, and a branch site (BS) upstream of the PPT-3Јss (Hastings and Krainer 2001;Black 2003). Spliceosome assembly is initiated by the binding of specific splicing factors to these signals: the U1 snRNP to the 5Јss, the protein SF1 to the BS, the U2 snRNP auxiliary factor U2AF large subunit (U2AF2; also known as U2AF65) to the PPT, and the U2AF small subunit (U2AF1; also known as U2AF35) to the 3Јss. In an ensuing reaction, U2 snRNP associates with the pre-mRNA through a base-pairing interaction between U2 snRNA (small nuclear RNA)...

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Section: Splicing In Y Lipolyticamentioning

confidence: 99%

Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes

Schwartz

Silva²,

Burstein³

et al. 2007

Genome Res.

166

174

View full text Add to dashboard Cite

show abstract

“…Another element in the upstream flank that may be distinct from an ISE is an anchoring site of 20 nt just upstream of the BP. This region has been implicated in sequence-independent binding of U2 snRNP proteins (Gozani et al 1996), but its role in splicing can be sequence dependent (Ast et al 2001). Finally, taking the extent of the polypyrimidine tract to be ‫41מ‬ is a bit arbitrary, as the overrepresentation of pyrimidines extends at a lower level beyond that distal limit (Penotti 1991;Stephens and Schneider 1992;.…”

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confidence: 99%

Dichotomous splicing signals in exon flanks

Zhang

Leslie

Chasin³

2005

Genome Res.

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Intronic elements flanking the splice-site consensus sequences are thought to play a role in pre-mRNA splicing. However, the generality of this role, the catalog of effective sequences, and the mechanisms involved are still lacking. Using molecular genetic tests, we first showed that the ∼50-nt intronic flanking sequences of exons beyond the splice-site consensus are generally important for splicing. We then went on to characterize exon flank sequences on a genomic scale. The G+C content of flanks displayed a bimodal distribution reflecting an exaggeration of this base composition in flanks relative to the gene as a whole. We divided all exons into two classes according to their flank G+C content and used computational and statistical methods to define pentamers of high relative abundance and phylogenetic conservation in exon flanks. Upstream pentamers were often common to the two classes, whereas downstream pentamers were totally different. Upstream and downstream pentamers were often identical around low G+C exons, and in contrast, were often complementary around high G+C exons. In agreement with this complementarity, predicted base pairing was more frequent between the flanks of high G+C exons. Pseudo exons did not exhibit this behavior, but rather tended to form base pairs between flanks and exon bodies. We conclude that most exons require signals in their immediate flanks for efficient splicing. G+C content is a sequence feature correlated with many genetic and genomic attributes. We speculate that there may be different mechanisms for splice site recognition depending on G+C content.[Supplemental material is available online at www.genome.org.]Pre-mRNA splicing is a fundamental step in gene expression. During this process, splice sites must be recognized within large introns before subsequent steps occur. How the splicing machinery manages to recognize splice sites remains a central problem in the study of splicing and its regulation. Three conserved sequence motifs, the 5Ј splice site, the 3Ј splice site and the branchpoint (BP), are known to be necessary for the two sequential transesterification reactions by which introns are removed and exons are joined (Adams et al. 1996;Jurica and Moore 2003). However, these sequences are quite degenerate; one can find about two orders of magnitude more intronic sequences that match either splice-site consensus, as well as or better than the sites that are actually used (Sun and Chasin 2000;Zhang et al. 2003). The BP consensus is even more degenerate Senapathy et al. 1990).There is strong genetic and biochemical evidence for the idea that the exon is the unit of initial recognition, and that there is coupling between the recognition of 5Ј and 3Ј splice sites across the exon (Robberson et al. 1990;Carothers et al. 1993;Berget 1995). However, even if pairing of 3' and 5' splice site-like sequences is stipulated and the distance between them constrained to a range typical for real exons, these "pseudo exons" still outnumber the real exons by at least an order of magnitu...

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“…Next, the U4/U5/U6 trisnRNP complex is added, resulting in an apparent destabilization of U1 snRNP from the spliceosome (reviewed in reference 24), followed by several rearrangements in which U1 is replaced by U5 and U6 at the 5Јss (1,4,5,22,27,32). The U4/U6 base pairing within the U4/U5/U6 complex is disrupted, U4 is released from the spliceosome, and U6 snRNA base pairs with U2 snRNA (3,10,26,36,37,60). These rearrangements finally allow the two constitutive catalytic steps to generate mature mRNA and liberate the intron.…”

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confidence: 99%

The U1 snRNP Base Pairs with the 5′ Splice Site within a Penta-snRNP Complex

Malca

Shomron

Ast

2003

Molecular and Cellular Biology

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Recognition of the 5 splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5 splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5 splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5 splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5 splice site through base pairing with the 5 end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5 splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5 splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5 splice site in a mammalian preassembled penta-snRNP complex.Splicing of precursor mRNA (pre-mRNA) is a critical regulatory stage in which accurate recognition and removal of introns by the splicing machinery compose the correct code for protein production. The splicing reaction is carried out by the spliceosome, a dynamic complex of five small nuclear ribonucleoproteins (snRNPs)-U1, U2, U4, U5, and U6-and many auxiliary proteins. The spliceosome assembles de novo on each intron and, through a myriad of RNA-RNA, RNAprotein, and protein-protein interactions, acts to excise each intron and ligate the exons (reviewed in references 11, 20, 24, and 45).Spliceosome assembly requires specific recognition of splice site signals: the 5Ј splice site (5Јss) consensus sequence, which includes a conserved GU dinucleotide at the 5Ј end of the intron; the 3Ј splice site (3Јss) region, which consists of the polypyrimidine tract and a conserved AG dinucleotide at the 3Ј end of the intron; and the branch site located ϳ20 nucleotides upstream of the 3Јss, bearing a conserved adenosine (8).According to the current model of spliceosome assembly on pre-mRNA, the snRNPs join the spliceosome in an ordered pathway. U1 snRNP initially recognizes the 5Јss by base pairing between U1 snRNA and the 5Јss exon-intron junction (at positions Ϫ3 to ϩ6); these are highly complementary sequences (17,35,48,50,64). Non-snRNP splicing factors interact with the 3Јss, resulting in the 5Јss being brought to the proximity of the 3Јss. The U1/5Јss base pairing is then weakened in an ATP-dependent step (9,21,23,29,30,53), allowing U2 snRNP to base pair with the branch site. Next, the U4/U5/U6 trisnRNP complex is added, resulting in an apparent destabilization of U1 snRNP from the spliceosome (reviewed in reference 24), followed by several rearrangements in which U1 is replaced by U5 and U6 at the 5Јss (1,4,5,22,27,32). The U4/U6 base pairing within the U4/U5...

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Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Cited by 15 publications

References 89 publications

Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes

Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes

Dichotomous splicing signals in exon flanks

The U1 snRNP Base Pairs with the 5′ Splice Site within a Penta-snRNP Complex

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