Multiple exon-binding sites in class II self-splicing introns
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Splice site mutation
Exon trapping
Exon shuffling
Exonic splicing enhancer
Splicing factor
We previously found that the splicing of exon 5 to exon 6 in the rat β-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al ., Genes and Dev. 2, 1627–1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro . We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro . We found that mutations in two regions of the pre-mRNA led to activation of the 3′-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3′-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5′-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3′-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5′-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3′-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5′-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro , such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5′-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.
Splice site mutation
Exon shuffling
Exon trapping
splice
Polypyrimidine tract
Precursor mRNA
Exonic splicing enhancer
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Intronic ratchet points (RPs) are abundant within long introns in the Drosophila genome and consist of juxtaposed splice acceptor and splice donor (SD) sites. Although they appear to encompass zero-nucleotide exons, we recently clarified that intronic recursive splicing (RS) requires a cryptic exon at the RP (an RS-exon), which is subsequently always skipped and thus absent from mRNA. In addition, Drosophila encodes a smaller set of expressed exons bearing features of RS. Here, we investigate mechanisms that regulate the choice between RP and RS-exon SDs. First, analysis of Drosophila RP SD mutants demonstrates that SD competition suppresses inclusion of cryptic exons in endogenous contexts. Second, characterization of RS-exon reporters implicates exonic sequences as influencing choice of RS-exon usage. Using RS-exon swap and mutagenesis assays, we show exonic sequences can determine RS-exon inclusion. Finally, we provide evidence that splicing can suppress utilization of RP SDs to enable RS-exon expression. Overall, multiple factors can influence splicing of Drosophila RS-exons, which usually result in their complete suppression as zero-nucleotide RPs, but occasionally yield translated RS-exons.
Exon trapping
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Exonic splicing enhancer
Splice site mutation
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splice
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During the pre-mRNA splicing process introns are removed and exons joined together in the resulting mature mRNA, which is then exported to the cytoplasm and translated into proteins. Several motifs in the nucleotide sequences near the exon-intron boundaries are required for proper exon definition including the 3’ and 5’ splice site consensus sequences (3’SS and 5’SS), which recruit basic splicing factors. In addition, auxiliary splicing regulatory elements, located in the upstream or downstream region of an exon, further influence exon recognition through the recruitment of additional binding proteins.
This study shows that intronic UG repeat elements in proximity of the 5’SS of an exon can function as splicing regulatory elements, generally enhancing the inclusion of the upstream exon in the final mRNA through the recruitment of UG repeats-binding proteins. In particular, the strength of the 5’SS consensus sequence affects this UG repeats-mediated splicing regulation. Furthermore, this study reveals that the UG repeats-binding protein TDP-43 acts as splicing modulator either activating of inhibiting the splicing events in different minigene systems. In fact, in presence of a disease-causing mutation at the 5’ end of the BRCA1 exon 12 TDP-43 enhances exon inclusion, acting as splicing enhancer. Alternatively, overexpression of TDP-43 can exert an inhibitory effect on splicing by promoting exon skipping in two newly identified TDP-43 target exons (RXRG exon 7 and ETF1 exon 7).
In conclusion, this study provides positive proof of concept that UG repeats located in the downstream region of poorly defined exons help 5’SS definition. Additionally, the study characterizes the role of TDP-43 in various splicing systems presenting the UG repeat-binding sites, with the practical application of evaluating a putative splicing-affecting pathological mutation.
Minigene
Exonic splicing enhancer
Exon shuffling
Splice site mutation
Exon skipping
Exon trapping
Polypyrimidine tract
SR protein
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Splice site mutation
Neurofibromin 1
Exon trapping
Exonic splicing enhancer
splice
Exon shuffling
Minigene
Exon skipping
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D M Helfman, W M Ricci, and L A Finn Cold Spring Harbor Laboratory, New York 11724.
Splice site mutation
Exon shuffling
Exon trapping
Minigene
Exonic splicing enhancer
Exon skipping
splice
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A 3 bp deletion located at the 5' end of exon 3 of MLH1, resulting in deletion of exon 3 from RNA, was recently identified.That this mutation disrupts an exon splicing enhancer (ESE) because it occurs in a purine-rich sequence previously identified as an ESE in other genes, and ESEs are often found in exons with splice signals that deviate from the consensus signals, as does the 3' splice signal in exon 3 of MLH1.The 3 bp deletion and several other mutations were created by polymerase chain reaction mutagenesis and tested using an in vitro splicing assay. Both mutant and wild type exon 3 sequences were cloned into an exon trapping vector and transiently expressed in Cos-1 cells.Analysis of the RNA indicates that the 3 bp deletion c.213_215delAGA (gi:28559089, NM_000249.2), a silent mutation c.216T-->C, a missense mutation c.214G-->C, and a nonsense mutation c.214G-->T all cause varying degrees of exon skipping, suggesting the presence of an ESE at the 5' end of exon 3. These mutations are situated in a GAAGAT sequence 3 bp downstream from the start of exon 3.The results of the splicing assay suggest that inclusion of exon 3 in the mRNA is ESE dependent. The exon 3 ESE is not recognised by all available motif scoring matrices, highlighting the importance of RNA analysis in the detection of ESE disrupting mutations.
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Splice site mutation
Exonic splicing enhancer
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Nonsense mutation
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Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy. In myotonic dystrophy, the lack of properly localized MBNL1 leads to missplicing of many pre-mRNAs. One of these events is the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3'-end of intron 4 is non-canonical in that it contains a predicted branch point that is 141 nucleotides from the 3'-splice site and an AAG 3'-splice site. Using a minigene that includes exon 4, intron 4, exon 5, intron 5, and exon 6 of MBNL1, we showed that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branch point confirmed that branching occurs primarily at the predicted distant branch point. Structure probing and footprinting revealed that the highly conserved region between the branch point and 3'-splice site is primarily unstructured and that MBNL1 binds within this region of the pre-mRNA. Deletion of the MBNL1 response element eliminated MBNL1 splicing regulation and led to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.
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Exon shuffling
Splice site mutation
Exonic splicing enhancer
Exon trapping
Group II intron
Polypyrimidine tract
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The cardiac troponin T (cTNT) pre-mRNA contains a single alternative exon (exon 5) which is either included or excluded from the processed mRNA. Using transient transfection of cTNT minigenes, we have previously localized pre-mRNA cis elements required for exon 5 alternative splicing to three small regions of the pre-mRNA which include exons 4, 5, and 6. In the present study, nucleotide substitutions were introduced into the region containing exon 5 to begin to define specific nucleotides required for exon 5 alternative splicing. A mutation within the 5' splice site flanking the cTNT alternative exon that increases its homology to the consensus sequence improves splicing efficiency and leads to increased levels of mRNAs that include the alternative exon. Surprisingly, substitution of as few as four nucleotides within the alternative exon disrupts cTNT pre-mRNA alternative splicing and prevents recognition of exon 5 as a bona fide exon. These results establish that the cTNT alternative exon contains information in cis that is required for its recognition by the splicing machinery.
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The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.
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The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.
Splice site mutation
Exon skipping
Exon shuffling
splice
Exon trapping
Exonic splicing enhancer
Minigene
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Polypyrimidine tract
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