Factors Affecting Authentic 5' Splice Site selection in Plant Nuclei
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To define elements critical for 5' splice selection in dicot plant nuclei, wild-type and mutant transcripts containing the first intron of the pea rbcS3A gene were expressed in vivo by using an autonomously replicating plant expression vector. Mutations within the normal 5' splice site (+1) of this intron demonstrate that 5' splice sites at the normal exon-intron boundary having only limited agreement with a 5' splice site consensus sequence can be spliced quite effectively in dicot nuclei. Inactivation of the normal 5' splice site occurs only by point mutations of the G at position +1 of the intron (+1G) or +2U or by multiple mutations at other positions and results in the activation of three cryptic 5' splice sites in the adjacent exon and intron. cis competition of cryptic sites having consensus 5' splice site sequences with the normal 5' splice site demonstrates that cryptic splice sites in the exon, but not the intron, can compete to some extent with the normal site. Replacement of the sequences between the cryptic and normal 5' splice sites with heterologous exon or intron sequences demonstrates that the 5' boundary of this plant intron is defined by its position relative to the AU transition point between exon and intron. These results suggest that potential 5' splice sites upstream of the AU transition point are accessible for recognition by the plant pre-mRNA splicing machinery and that those downstream in the AU-rich intron are masked from recognition.Keywords:
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Journal Article Splice site selection by intron a13 of the COX1 gene from Saccharomyces cerevisiae Get access Arend J. Winter, Arend J. Winter EC Slater Institute for Biochemical Research, Academisch Medisch Centrum, University of AmsterdamMeibergdreef 15, 1105 AZ Amsterdam, The Netherlands + Present address: Institute de Biologie Moleèculaire et Cellulaire du CNRS, 15 Rue Reneè Descartes, Strasbourg, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Marian J.A. Groot Koerkamp, Marian J.A. Groot Koerkamp EC Slater Institute for Biochemical Research, Academisch Medisch Centrum, University of AmsterdamMeibergdreef 15, 1105 AZ Amsterdam, The Netherlands Search for other works by this author on: Oxford Academic PubMed Google Scholar Henk F. Tabak Henk F. Tabak * EC Slater Institute for Biochemical Research, Academisch Medisch Centrum, University of AmsterdamMeibergdreef 15, 1105 AZ Amsterdam, The Netherlands * To whom correpondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 20, Issue 15, 11 August 1992, Pages 3897–3904, https://doi.org/10.1093/nar/20.15.3897 Published: 11 August 1992 Article history Received: 27 May 1992 Received: 09 July 1992 Received: 09 July 1992 Published: 11 August 1992
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Expressed sequence tag (EST) databases represent a large volume of information on expressed genes including tissue type, expression profile and exon structure. In this study we create an extensive data set of human alternative splicing. We report the analysis of 7867 non‐redundant mRNAs, 3011 of which contained alternative splice forms (38% of all mRNAs analysed). From a total of 12 572 ESTs 4560 different possible alternative splice forms were detected. Interestingly, 70% of the alternative splice forms correspond to exon deletion events with only 30% exonic insertions. We experimentally verified 19 different splice forms from 16 genes in a total subset of 20 studied; all of the respective genes are of medical relevance.
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Splicing of small introns in lower eucaryotes can be distinguished from vertebrate splicing by the inability of such introns to be expanded and by the inability of splice site mutations to cause exon skipping—properties suggesting that the intron rather than the exon is the unit of recognition. Vertebrates do contain small introns. To see if they possess properties similar to small introns in lower eucaryotes, we studied the small second intron from the human α-globin gene. Mutation of the 5′ splice site of this intron resulted in in vivo intron inclusion, not exon skipping, suggesting the presence of intron bridging interactions. The intron had an unusual base composition reflective of a sequence bias present in a collection of small human introns in which multiple G triplets stud the interior of the introns. Each G triplet represented a minimal sequence element additively contributing to maximal splicing efficiency and spliceosome assembly. More importantly, G triplets proximal to a duplicated splice site caused preferential utilization of the 5′ splice site upstream of the triplets or the 3′ splice site downstream of the triplets; i.e., sequences containing G triplets were preferentially used as introns when a choice was possible. Thus, G triplets internal to a small intron have the ability to affect splice site decisions at both ends of the intron. Each G triplet additively contributed to splice site selectivity. We suggest that G triplets are a common component of human 5′ splice sites and aid in the definition of exon-intron borders as well as overall splicing efficiency. In addition, our data suggest that such intronic elements may be characteristic of small introns and represent an intronic equivalent to the exon enhancers that facilitate recognition of both ends of an exon during exon definition.
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Limiting
ENCODE
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In vivo analyses of cis-acting sequence requirements for pre-mRNA splicing in tobacco nuclei have previously demonstrated that the 5' splice sites are selected by their position relative to AU-rich elements within plant introns and by their degree of complementarity to the U1 small nuclear RNA. To determine whether the presence of adjacent introns affects 5' splice site recognition in plant nuclei, we have analyzed the in vivo splicing patterns of two-intron constructs containing 5' splice site mutations in the second intron. These experiments indicated that the splice site selection patterns in plant nuclei are defined primarily by sequences within the intron (intron definition) and secondarily by weak interactions across exons (exon definition). The effects of these secondary interactions became evident only when mutations in the downstream 5' splice site decreased its functionality and differed depending on the availability of cryptic splice sites close to the mutant site. In beta-conglycinin chimeric transcripts containing multiple cryptic 5' splice sites, the presence of an intact upstream intron significantly increased splicing at the downstream 5' splice sites in a polar fashion without activating exon skipping. In a natural beta-conglycinin transcript, which does not contain cryptic 5'splice sites, mutation of the first nucleotide of the downstream intron activated an array of noncanonical 5' and 3' splice sites and some exon skipping.
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In vertebrates most exons are small surrounded by much larger introns. Due to this architecture, splice sites are initially recognized across the exon in what is known as exon definition. Here we examine the relative usage of 3,000 pairs of alternative 3′ splice sites and 2,000 pairs of alternative 5′ splice sites in the human genome. We find that within an optimal exon length window >50 <250 nts, there is a positive correlation between splice site strength and splice site usage. However, when one splice site is located inside of this exon length and the other is not, the splice site inside of the optimal exon length is preferred regardless of splice site strength. Our analysis reveals that splice sites within an optimal exon size are preferentially used when compared to splice sites outside of this optimal size. We conclude that a constraint of exon size is a fundamental parameter that dictates splice site usage. Because the spliceosome eventually forms across the intron, it is believed that intron definition is the predominant mode of splice site recognition for introns smaller than 250 nucleotides in length. However, in a subset of our alternative splice sites, in which one splice site is exon defined and the other is intron defined, the exon defined splice site is preferentially used, suggesting that exon recognition is the default mode of splice site recognition in vertebrates. NIH
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TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.
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The exon definition model accounts for the paring between the 3’ splice site and its downstream 5’splice site [5]. It was demonstrated experimentally that 5’ and 3’ splice-site pairing in metazoanpre-mRNAs occurred in the two distinct steps [1]. In our previous paper, we proposed a subclassmethod to predict 5’ splice sites in mammalian pre-mRNAs, and suggested that several 5’ splice sitepattern sequences were observed more frequently on the first or second introns than on the otherorder introns [3]. However, the relation between the 5’ splice site sequences and the order of introns iscurrently under investigation. On the other hand, positional characterization of false positives usingcomputational prediction of human splice sites was presented [6]. In the present study, by using aposition-tree method to analyze the distribution of the 5’ splice site-like sequences in human andmouse pre-mRNAs, we obtained the 5’ splice site sequences whose lengths were minimal but weresufficient for specifying the 5’ splice sites. Then we investigated the distribution of the 5’ splice site-like sequences that were one nucleotide shorter than the obtained 5’ splice site sequences. As a result,it was confirmed that the 5’ splice site-like sequences had the tendency of the uneven distributionwithin pre-mRNAs.
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We have carried out an initial analysis of the dynamics of the recent evolution of the splice-sites sequences on a large collection of human, rodent (mouse and rat), and chicken introns. Our results indicate that the sequences of splice sites are largely homogeneous within tetrapoda. We have also found that orthologous splice signals between human and rodents and within rodents are more conserved than unrelated splice sites, but the additional conservation can be explained mostly by background intron conservation. In contrast, additional conservation over background is detectable in orthologous mammalian and chicken splice sites. Our results also indicate that the U2 and U12 intron classes seem to have evolved independently since the split of mammals and birds; we have not been able to find a convincing case of interconversion between these two classes in our collections of orthologous introns. Similarly, we have not found a single case of switching between AT-AC and GT-AG subtypes within U12 introns, suggesting that this event has been a rare occurrence in recent evolutionary times. Switching between GT-AG and the noncanonical GC-AG U2 subtypes, on the contrary, does not appear to be unusual; in particular, T to C mutations appear to be relatively well tolerated in GT-AG introns with very strong donor sites.
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