Wobble Splicing Reveals the Role of the Branch Point Sequence-to-NAGNAG Region in 3′ Tandem Splice Site Selection

Tarn, Woan‐Yuh; Lin, Wen-chang

doi:10.1128/mcb.00363-07

Cited by 25 publications

(37 citation statements)

References 42 publications

(47 reference statements)

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“…1B). This finding was consistent with a report by Tsai et al (2007), which demonstrated that the "N" nucleotide preceding AG plays a critical role in the 3' wobble splicing and the ratio (up to 87.9%) of distal AG selection in NAGNAG tandem motif. In addition, the KIT receptor is characterized by the presence of five immunoglobulin-like domains in its extracellular portion (Liu et al, 2007).…”

Section: Resultssupporting

confidence: 94%

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

et al. 2011

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The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.

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Section: Resultssupporting

confidence: 94%

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

et al. 2011

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“…At some minimal distance, spatial constraints of the spliceosome will likely dictate limits on the branchpoints and splice sites used in the excision of the intron. The contribution to 3 ′ splice site choice that the physical distance from the alternative 3 ′ splice site to the branchpoint makes has been explored in other organisms (Akerman and MandelGutfreund 2006;Tsai et al 2007Tsai et al , 2010Bradley et al 2012). Expression analysis of a PTB minigene with 4-nt or 7-nt insertions between the branchpoint and tandem acceptor set caused an increase in splicing to the proximal site, suggesting that the choice of branchpoint may influence the choice of 3 ′ splice site at NAGNAGs (Tsai et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites inC. elegans

et al. 2015

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Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3 ′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3 ′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3 ′ splice site (furthest from the 5 ′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3 ′ splice site (closer to the 5 ′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3 ′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3 ′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3 ′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. [Supplemental material is available for this article.]Alternative splicing is a highly regulated process by which a cell can produce multiple messenger RNAs, potentially encoding multiple proteins, from a common precursor transcript. The de novo assembly of a spliceosome on each intron of a pre-mRNA transcript requires cis-elements within the intron that are recognized as signals marking the beginning, end, and branchpoint (Ares and Weiser 1995). A cassette exon is a form of alternative splicing in which an exon is either included or skipped in the mature mRNA. Conserved enhancer or silencer elements within the exon or the surrounding introns interact with an array of constitutive and tissue-specific trans-factors that promote or inhibit assembly of a functional spliceosome (Wang and Burge 2008). The use of alternative 3 ′ or 5 ′ splice sites modifies gene expression by including or skipping coding sequences at the ends of exons. Many examples of adjacent alternative 3 ′ splice sites, defined as being separated by 18 nucleotides (nt) or less, have been observed. In many species, a form of alternative 3 ′ splice site usage has been identified in which the alternative splicing acceptors are only 3 nt apart. Except for the rare example of AC dinucleotides observed for substrates of the minor spliceosome, introns end with AG dinucleotides. Alternative 3 ′ splice sites separated by only 3 nt are referred to as NAGNAGs, as the end of the intron consists of two AG splice acceptors separated by 3 nt. A...

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“…In addition, NAGNAG acceptors are very common in genes from a variety of organisms from land plants to humans (Hiller et al 2004;Schindler et al 2008). Although the use of the alternative NAGNAG sites (known as 39 splice junction wobbling) has been argued to be stochastic (Chern et al 2006;Sinha et al 2009), evidence exists for functional effects of the single amino acid differences generated by the use of the alternative acceptors (Tsai et al 2008) and for arrangements of regulatory sequences at these regions (Akerman and Mandel-Gutfreund 2006;Tsai et al 2007). …”

Section: Splicing Defect In Alps Mutationmentioning

confidence: 99%

Strict 3′ splice site sequence requirements for U2 snRNP recruitment after U2AF binding underlie a genetic defect leading to autoimmune disease

Corrionero

Raker²,

Izquierdo³

et al. 2011

RNA

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We report that the 39 splice site associated with the alternatively spliced exon 6 of the Fas receptor CD95 displays strict sequence requirements and that a mutation that disrupts this particular sequence arrangement leads to constitutive exon 6 skipping in a patient suffering from autoimmune lymphoproliferative syndrome (ALPS). Specifically, we find an absolute requirement for RCAG/G at the 39 splice site (where R represents purine, and / indicates the intron/exon boundary) and the balance between exon inclusion and skipping is exquisitely sensitive to single nucleotide variations in the uridine content of the upstream polypyrimidine (Py)-tract. Biochemical experiments revealed that the ALPS patient mutation reduces U2 snRNP recruitment to the 39 splice site region and that this effect cannot be explained by decreased interaction with the U2 snRNP Auxiliary Factor U2AF, whose 65-and 35-kDa subunits recognize the Py-tract and 39 splice site AG, respectively. The effect of the mutation, which generates a tandem of two consecutive AG dinucleotides at the 39 splice site, can be suppressed by increasing the distance between the AGs, mutating the natural 39 splice site AG or increasing the uridine content of the Py-tract at a position distal from the 39 splice site. The suppressive effects of these additional mutations correlate with increased recruitment of U2 snRNP but not with U2AF binding, again suggesting that the strict architecture of Fas intron 5 39 splice site region is tuned to regulate alternative exon inclusion through modulation of U2 snRNP assembly after U2AF binding.

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Wobble Splicing Reveals the Role of the Branch Point Sequence-to-NAGNAG Region in 3′ Tandem Splice Site Selection

Cited by 25 publications

References 42 publications

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites inC. elegans

Strict 3′ splice site sequence requirements for U2 snRNP recruitment after U2AF binding underlie a genetic defect leading to autoimmune disease

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