Prp8 impacts cryptic but not alternative splicing frequency

Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5′ splice site, 3′ splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or “cryptic” splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed aCaenorhabditis elegansgenetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome’s catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of theseprp-8mutantC. elegansreveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.

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Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.966-972.1984 ◽

1984 ◽

Vol 4 (5) ◽

pp. 966-972

Author(s):

C Montell ◽

E F Fisher ◽

M H Caruthers ◽

A J Berk

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Early Phase ◽

Late Phase ◽

Productive Infection ◽

Splice Sites ◽

Mrna Synthesis ◽

Adenovirus E1b ◽

Cryptic Splice Sites ◽

Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

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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

Neuromuscular Disorders ◽

10.1016/j.nmd.2009.06.108 ◽

2009 ◽

Vol 19 (8-9) ◽

pp. 577

Author(s):

M. Matsuo ◽

Y. Habara ◽

Y. Takeshima ◽

H. Awano ◽

Y. Okizuka ◽

...

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Dystrophin Gene ◽

Cryptic Splice Site ◽

In Vitro Splicing ◽

Splicing Patterns

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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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hnRNP A1 Regulates Alternative Splicing of Tau Exon 10 by Targeting 3′ Splice Sites

Cells ◽

10.3390/cells9040936 ◽

2020 ◽

Vol 9 (4) ◽

pp. 936 ◽

Cited By ~ 1

Author(s):

Yongchao Liu ◽

Donggun Kim ◽

Namjeong Choi ◽

Jagyeong Oh ◽

Jiyeon Ha ◽

...

Keyword(s):

Gene Expression ◽

Gene Ontology ◽

Alternative Splicing ◽

Splice Site ◽

Hnrnp A1 ◽

Gene Ontology Analysis ◽

Splice Sites ◽

Neuronal Function ◽

Brain Functions ◽

Exon 10

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3′ splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.

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The Sex-lethal early splicing pattern uses a default mechanism dependent on the alternative 5' splice sites.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1674 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1674-1681 ◽

Cited By ~ 8

Author(s):

C Zhu ◽

J Urano ◽

L R Bell

Keyword(s):

Tissue Culture ◽

Splice Site ◽

Site Preference ◽

Splice Sites ◽

Culture Cells ◽

Tissue Culture Cells ◽

Important Region ◽

Splicing Pattern ◽

Sex Lethal ◽

Splicing Patterns

The Sex-lethal (Sxl) early transcripts have a unique 5' exon and a splicing pattern that differs from that of the late transcripts. While the late transcripts are regulated sex specifically by control of exon 3 inclusion, the early transcripts are not. While the late transcripts include exon 3 by default, the early transcripts skip exon 3. Splicing patterns of a reporter gene that mimics the early transcript, and its variants, were analyzed in Drosophila transformants and tissue culture cells. The results demonstrate that the early, in contrast to the late, splicing pattern is not regulated by stage-specific or sex-specific trans-acting factors, and so the pattern appears to arise from some type of intrinsic splice site preference or compatibility. Inclusion or exclusion of exon 3 is determined by the identity of the upstream 5' splice site region as late or early. The important region of the early exon lies within 233 nucleotides of the immediately adjacent intron.

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Combinatorial Control of Exon Recognition

Journal of Biological Chemistry ◽

10.1074/jbc.r700035200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1211-1215 ◽

Cited By ~ 144

Author(s):

Klemens J. Hertel

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Cell Types ◽

Splice Sites ◽

Splice Site Selection ◽

Multiple Parameters ◽

Splicing Regulators ◽

Eukaryotic Genes ◽

Different Cell Types ◽

Mature Mrnas

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which catalyzes the removal of noncoding intronic sequences to assemble exons into mature mRNAs prior to export and translation. Given the complexity of higher eukaryotic genes and the relatively low level of splice site conservation, the precision of the splicing machinery in recognizing and pairing splice sites is impressive. Introns ranging in size from <100 up to 100,000 bases are removed efficiently. At the same time, a large number of alternative splicing events are observed between different cell types, during development, or during other biological processes. This extensive alternative splicing implies a significant flexibility of the spliceosome to identify and process exons within a given pre-mRNA. To reach this flexibility, splice site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice site strength, the presence or absence of splicing regulators, RNA secondary structures, the exon/intron architecture, and the process of pre-mRNA synthesis itself. The relative contributions of each of these parameters control how efficiently splice sites are recognized and flanking introns are removed.

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Factor interactions with the simian virus 40 early pre-mRNA influence branch site selection and alternative splicing

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2007-2017.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2007-2017

Author(s):

J C Noble ◽

H Ge ◽

M Chaudhuri ◽

J L Manley

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Rnase Protection Assay ◽

Simian Virus 40 ◽

Simian Virus ◽

Splice Sites ◽

Rnase Protection ◽

Branch Site ◽

Factor Interactions ◽

Selection Of

To study the interaction of splicing factors with the simian virus 40 early-region pre-RNA, which can be alternatively spliced to produce large T and small t mRNAs, we used an in vitro RNase protection assay that defines the 5' boundaries of factor-RNA interactions. Protection products reflecting factor interactions with the large T and small t 5' splice sites and with the multiple lariat branch site region were characterized. All protection products were detected very early in the splicing reaction, before the appearance of spliced RNAs. However, protection of the large T 5' splice site was detected well before small t 5' splice site and branch site protection products, which appeared simultaneously. Oligonucleotide-targeted degradation of small nuclear RNAs (snRNAs) revealed that protection of the branch site region, which occurred at multiple sites, required intact U2 snRNA and was enhanced by U1 snRNA, while protection of the large T and small t 5' splice sites required both U1 and U2 snRNAs. Analysis of several pre-RNAs containing mutations in the branch site region suggests that factor interactions involving the multiple copies of the branch site consensus determine the selection of branch points, which is an important factor in the selection of alternative splicing pathways.

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An Apparent Pseudo-Exon Acts both as an Alternative Exon That Leads to Nonsense-Mediated Decay and as a Zero-Length Exon

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2237-2246.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2237-2246 ◽

Cited By ~ 26

Author(s):

Sushma-Nagaraja Grellscheid ◽

Christopher W. J. Smith

Keyword(s):

Splice Site ◽

Alternative Exon ◽

Splice Sites ◽

Exon 2 ◽

Nonsense Mediated Decay ◽

Exon Splicing ◽

Exon 4 ◽

Cryptic Splice Sites

ABSTRACT Pseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat α-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when “prespliced” to exon 2 it follows a “zero length exon” splicing pathway in which a newly generated 5′ splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD.

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Characterisation and quantification of F8 transcripts of ten putative splice site mutations

Thrombosis and Haemostasis ◽

10.1160/th14-06-0523 ◽

2015 ◽

Vol 113 (03) ◽

pp. 585-592 ◽

Cited By ~ 2

Author(s):

Yeling Lu ◽

Yufeng Ruan ◽

Qiulan Ding ◽

Xuefeng Wang ◽

Xiaodong Xi ◽

...

Keyword(s):

Splice Site ◽

Mrna Level ◽

Gene Mutations ◽

Exon Skipping ◽

Splice Sites ◽

Wild Type ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Fluorescent Pcr ◽

Cryptic Splice Sites

SummaryMutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %>, 14.2 %>, 5.2 %>, 19.2 %>, 1.8 °% and 2.5 %> of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

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