T.P.1.03 In vitro splicing analysis reveals that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G>A mutations in introns of the dystrophin gene

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.

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Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.13.5902-5910.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 5902-5910 ◽

Cited By ~ 13

Author(s):

Zhi-Ming Zheng ◽

Jesse Quintero ◽

Eric S. Reid ◽

Christian Gocke ◽

Carl C. Baker

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Splicing Factors ◽

Bovine Papillomavirus ◽

Splicing Pattern ◽

In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

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Primary structure of rat plasma membrane Ca2+-ATPase isoform 4 and analysis of alternative splicing patterns at splice site A

Biochemical Journal ◽

10.1042/bj3060779 ◽

1995 ◽

Vol 306 (3) ◽

pp. 779-785 ◽

Cited By ~ 45

Author(s):

T P Keeton ◽

G E Shull

Keyword(s):

Plasma Membrane ◽

Alternative Splicing ◽

Splice Site ◽

Primary Structure ◽

Sequence Divergence ◽

Rat Plasma ◽

Pcr Analysis ◽

Rat Tissues ◽

Mrna Tissue Distribution ◽

Splicing Patterns

We have determined the primary structure of the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4), and have analysed its mRNA tissue distribution and alternative splicing patterns at splice site A. Rat PMCA4 (rPMCA4) genomic clones were isolated and used to determine the coding sequences and intron/exon organization of the 5′-end of the gene, and the remaining coding sequence was determined from PCR-amplified cDNA fragments. Pairwise comparisons reveal that the amino acid sequence of rPMCA4 has diverged substantially from those of rPMCA isoforms 1, 2 and 3 (73-76% identity) and from that of human PMCA4 (87%). Despite the high degree of sequence divergence between the two species, comparisons of intron and untranslated mRNA sequences with the corresponding human sequences confirm the identity of this rat isoform as PMCA4. Northern blot studies demonstrate that the PMCA4 mRNA is expressed in all rat tissues examined except liver, with the highest levels in uterus and stomach. A combination of PCR analysis of alternative splicing patterns and sequence analysis of the gene demonstrate that a 36 nt exon at site A is included in PMCA4 mRNAs of most tissues but is largely excluded in heart and testis. Alternative splicing of both the 36 nt exon and a previously characterized 175 nt exon at splice site C, each of which can be either included or excluded in a highly tissue-specific manner, leads to the production of four different PMCA4 variants ranging in size from 1157 to 1203 amino acids.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2774 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2774-2780 ◽

Cited By ~ 20

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3582 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3582-3590 ◽

Cited By ~ 21

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2993-3001.1993 ◽

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.

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The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4852 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4852-4861 ◽

Cited By ~ 35

Author(s):

K Chebli ◽

R Gattoni ◽

P Schmitt ◽

G Hildwein ◽

J Stevenin

Keyword(s):

Splice Site ◽

Site Directed Mutagenesis ◽

Hairpin Structure ◽

Cis Acting ◽

Adenovirus E1a ◽

Sequence Elements ◽

In Vitro Splicing ◽

Hairpin Formation

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.

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The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.860 ◽

1988 ◽

Vol 8 (2) ◽

pp. 860-866 ◽

Cited By ~ 20

Author(s):

P J Furdon ◽

R Kole

Keyword(s):

Splice Site ◽

Mrna Splicing ◽

Beta Globin ◽

Specific Sequence ◽

In Vitro Splicing ◽

Intron 2 ◽

Specific Sequences

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.

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SRp30c Is a Repressor of 3′ Splice Site Utilization

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4001-4010.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4001-4010 ◽

Cited By ~ 43

Author(s):

Martin J. Simard ◽

Benoit Chabot

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Binding Sites ◽

Nuclear Protein ◽

Cross Linking ◽

Dependent Manner ◽

Hnrnp A1 ◽

High Affinity ◽

Specific Association

ABSTRACT Several intron elements influence exon 7B skipping in the mammalian hnRNP A1 pre-mRNA. We have shown previously that the 38-nucleotide CE9 element located in the intron separating alternative exon 7B from exon 8 can repress the use of a downstream 3′ splice site. The ability of CE9 to act on heterologous substrates, combined with the results of competition and gel shift assays, indicates that the activity of CE9 is mediated by a trans-acting factor. UV cross-linking analysis revealed the specific association of a 25-kDa nuclear protein with CE9. Using RNA affinity chromatography, we isolated a 25-kDa protein that binds to CE9 RNA. This protein corresponds to SRp30c. Consistent with a role for SRp30c in the activity of CE9, recombinant SRp30c interacts specifically with CE9 and can promote splicing repression in vitro in a CE9-dependent manner. The closest homologue of SRp30c, ASF/SF2, does not bind to CE9 and does not repress splicing even when the intronic SRp30c binding sites are replaced with high-affinity ASF/SF2 binding sites. Only the first 7 nucleotides of CE9 are sufficient for binding to SRp30c, and mutations that abolish binding also prevent repression. Our results indicate that SRp30c can function as a repressor of 3′ splice site utilization and suggest that the SRp30c-CE9 interaction may contribute to the control of hnRNP A1 alternative splicing.

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