The genomic organization of the human corticotropin-releasing factor type-1 receptor
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Coding region
Exon shuffling
The human alpha-tropomyosin gene hTMnm has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.
Minigene
Exon shuffling
Exon trapping
Splice site mutation
Tropomyosin
Exonic splicing enhancer
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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
Minigene
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RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts
Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein-protein interactions.
Exon trapping
Exonic splicing enhancer
Exon shuffling
Splice site mutation
Exon skipping
Splicing factor
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Exon shuffling
Exon trapping
Coding region
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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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Polypyrimidine tract
Exon shuffling
Exon trapping
Splice site mutation
Exonic splicing enhancer
splice
Exon skipping
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Citations (21)
The rat β-tropomyosin (β-TM) gene encodes both skeletal muscle β-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1–5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3′ splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5′ splice site of exon 7 in nonmuscle cells. The 5′ splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5′ splice site of exon 7 does not result from the sequences at the 3′ end of intron 6 that block the use of the 3′ splice site of exon 7. However, mutating two conserved nucleotides GU at the 5′ splice site of exon 6 results in the efficient use of the 5′ splice site of exon 7. In addition, a mutation that changes the 5′ splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5′ splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.
Splice site mutation
Exon shuffling
splice
Exon trapping
Exon skipping
Minigene
Exonic splicing enhancer
Polypyrimidine tract
Precursor mRNA
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The human alpha-tropomyosin gene hTMnm has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.
Minigene
Exon shuffling
Exon trapping
Splice site mutation
Tropomyosin
Exonic splicing enhancer
Exon skipping
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TTN is a major disease‐causing gene in cardiac muscle. TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by alternative exon usages of exon 45 (Novex 1), exon 46 (Novex 2) and exon 48 (Novex 3). It is unknown how these exons are alternatively used in Novex isoforms across species and whether Novex isoforms are associated with heart disease. In this study, we compared alternative exon usage between species with the mVISTA online tool, and found that exon 45 is conserved across all species, but exons 46 and 48 only found in some species. Interestingly, we found a conserved region between exons 47 and 48 across species which has never been characterized before. RT‐PCR and DNA sequencing confirmed that this is a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, we found two new splicing forms, which one has intron 44 retention and the other has intron 45 retention. With Novex 2 primer pairs, we found that Novex 2 is lack of expression in pig, mouse and rat. A new truncated isoform with intron 46 retention is mainly expressed in human and frog heart, and another new truncated isoform with exon 45 and 46 are co‐expressed predominantly in chicken and frog heart. Using RBM20 knockout heart, we revealed that RBM20 doesn't regulate splicing of Novex isoforms. Finally, we examined the expression levels of Novex isoforms in human heart with cardiomyopathies, and we didn't find any difference. These results suggest that exon usage of Novex isoforms varies across species which is not associated with cardiomyopathies. Hence, their functional role in the heart need be further studied in the future, which is beyond the scope of this study. Support or Funding Information This work was supported by the NIFA‐USDA 1009266, National Institutes of Health NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of Wyoming. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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Coding region
Exon shuffling
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