scholarly journals Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicity in vitro

2019 ◽  
Vol 6 (8) ◽  
pp. 191128 ◽  
Author(s):  
Jonathan Cook ◽  
Elizabeth de Wolf ◽  
Nicholas Dale
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Future Research ◽  
Cryptic Splice Site ◽  
Toxicity In Vitro

The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO ­2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

scholarly journals Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

1988 ◽  
Vol 8 (9) ◽  
pp. 3582-3590 ◽  
Author(s):  
X Y Fu ◽  
J D Colgan ◽  
J L Manley
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Sequence ◽  
Small T Antigen

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells.

Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA

1988 ◽  
Vol 8 (9) ◽  
pp. 3582-3590
Author(s):  
X Y Fu ◽  
J D Colgan ◽  
J L Manley
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Sequence ◽  
Small T Antigen

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells.

scholarly journals A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Oncogenesis ◽  
2021 ◽  
Vol 10 (5) ◽  
Author(s):  
Eleanor Star ◽  
Megan Stevens ◽  
Clare Gooding ◽  
Christopher W. J. Smith ◽  
Ling Li ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Fluorescent Protein ◽  
Drug Repositioning ◽  
Egfp Expression ◽  
In Vivo Model ◽  
Terminal Exon ◽  
Angiogenic Activity

AbstractAlternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

scholarly journals Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.

1997 ◽  
Vol 17 (5) ◽  
pp. 2774-2780 ◽  
Author(s):  
C F Kennedy ◽  
S M Berget
Keyword(s):  
Drosophila Melanogaster ◽  
Branch Point ◽  
Splice Site ◽  
Minimum Size ◽  
Cross Linking ◽  
In Vitro Splicing ◽  
Precursor Rna ◽  
Minimal Region

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

scholarly journals Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF

1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factor ◽  
Alternative Exon ◽  
Hnrnp A1 ◽  
Exon Inclusion ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

scholarly journals Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract

1999 ◽  
Vol 73 (3) ◽  
pp. 2394-2400 ◽  
Author(s):  
Craig R. Cook ◽  
Mark T. McNally
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Rna Splicing ◽  
Nuclear Extract ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
A Minor

ABSTRACT The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several ciselements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.

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