Sequence requirements in different steps of the pre-mRNA splicing reaction: analysis by the RNA modification-exclusion technique

1990 ◽  
Vol 10 (9) ◽  
pp. 4942-4947
Author(s):  
K M Lang ◽  
W Keller
Keyword(s):  
Complex Formation ◽  
Splice Site ◽  
Point Mutations ◽  
Mrna Splicing ◽  
Rna Modification ◽  
Modified Nucleotides ◽  
Complete Set ◽  
Stepwise Assembly ◽  
Sequence Requirements ◽  
Single Reaction

The stepwise assembly of splicing complexes and the subsequent splicing reaction were analyzed by the RNA modification-exclusion technique, which generates the equivalent of a complete set of point mutations in a single reaction. We found that although the sequences surrounding the 5' splice site, the branch point, and the 3' splice site, including the 3' AG, were required for presplicing complex formation, modified nucleotides at these positions were not completely excluded. The same sequences were required for splicing complex formation; however, modified nucleotides in these sequences were excluded to a much greater extent.

scholarly journals Sequence requirements in different steps of the pre-mRNA splicing reaction: analysis by the RNA modification-exclusion technique.

1990 ◽  
Vol 10 (9) ◽  
pp. 4942-4947 ◽  
Author(s):  
K M Lang ◽  
W Keller
Keyword(s):  
Complex Formation ◽  
Splice Site ◽  
Point Mutations ◽  
Mrna Splicing ◽  
Rna Modification ◽  
Modified Nucleotides ◽  
Complete Set ◽  
Stepwise Assembly ◽  
Sequence Requirements ◽  
Single Reaction

The stepwise assembly of splicing complexes and the subsequent splicing reaction were analyzed by the RNA modification-exclusion technique, which generates the equivalent of a complete set of point mutations in a single reaction. We found that although the sequences surrounding the 5' splice site, the branch point, and the 3' splice site, including the 3' AG, were required for presplicing complex formation, modified nucleotides at these positions were not completely excluded. The same sequences were required for splicing complex formation; however, modified nucleotides in these sequences were excluded to a much greater extent.

scholarly journals Mutagenesis of the Yeast Gene PRP8 Reveals Domains Governing the Specificity and Fidelity of 3′ Splice Site Selection

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 723-739 ◽  
Author(s):  
James G Umen ◽  
Christine Guthrie
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Point Mutations ◽  
Genetic Data ◽  
Mrna Splicing ◽  
Terminal Portion ◽  
Yeast Gene ◽  
Splice Site Selection ◽  
Snrnp Protein ◽  
New Alleles

Abstract PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the undine tract that precedes most yeast 3′ slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3′ splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3′ splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the pp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3′ splice site selection and fidelity of 3′ splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing.

Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1345-1353
Author(s):  
Amber K Bowers ◽  
Jennifer A Keller ◽  
Susan K Dutcher
Keyword(s):  
Molecular Markers ◽  
Chlamydomonas Reinhardtii ◽  
Candidate Genes ◽  
Splice Site ◽  
Genomic Dna ◽  
Ribosomal Proteins ◽  
Genomic Sequence ◽  
Point Mutations ◽  
Acceptor Site ◽  
Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

scholarly journals A 5′ splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated proα1(I) chains with a non-collagenous insertion destabilizing the triple helix

1994 ◽  
Vol 302 (3) ◽  
pp. 729-735 ◽  
Author(s):  
J F Bateman ◽  
D Chan ◽  
I Moeller ◽  
M Hannagan ◽  
W G Cole
Keyword(s):  
Splice Site ◽  
De Novo ◽  
Donor Site ◽  
Mrna Splicing ◽  
Splice Donor Site ◽  
Sequence Motif ◽  
Type I ◽  
Splice Donor ◽  
Site Mutation ◽  
Definition Of

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3′ limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.

Role of the 3′ splice site consensus sequence in mammalian pre-mRNA splicing

Nature ◽  
1985 ◽  
Vol 317 (6039) ◽  
pp. 732-734 ◽  
Author(s):  
Barbara Ruskin ◽  
Michael R. Green
Keyword(s):  
Splice Site ◽  
Consensus Sequence ◽  
Mrna Splicing ◽  
Site Consensus

scholarly journals U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

1993 ◽  
Vol 13 (5) ◽  
pp. 2666-2676 ◽  
Author(s):  
J B Cohen ◽  
S D Broz ◽  
A D Levinson
Keyword(s):  
Splice Site ◽  
Mammalian Cells ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
Gene Complementation ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

scholarly journals A plugin for the Ensembl Variant Effect Predictor that uses MaxEntScan to predict variant spliceogenicity

2018 ◽  
Vol 35 (13) ◽  
pp. 2315-2317 ◽  
Author(s):  
Jannah Shamsani ◽  
Stephen H Kazakoff ◽  
Irina M Armean ◽  
Will McLaren ◽  
Michael T Parsons ◽  
...  
Keyword(s):  
Splice Site ◽  
Disease Risk ◽  
Cancer Susceptibility ◽  
Mrna Splicing ◽  
Supplementary Information ◽  
Mendelian Disease ◽  
Functional Protein ◽  
Variant Effect Predictor ◽  
Cancer Susceptibility Genes ◽  
Variant Effect

Abstract Summary Assessing the pathogenicity of genetic variants can be a complex and challenging task. Spliceogenic variants, which alter mRNA splicing, may yield mature transcripts that encode non-functional protein products, an important predictor of Mendelian disease risk. However, most variant annotation tools do not adequately assess spliceogenicity outside the native splice site and thus the disease-causing potential of variants in other intronic and exonic regions is often overlooked. Here, we present a plugin for the Ensembl Variant Effect Predictor that packages MaxEntScan and extends its functionality to provide splice site predictions using a maximum entropy model. The plugin incorporates a sliding window algorithm to predict splice site loss or gain for any variant that overlaps a transcript feature. We also demonstrate the utility of the plugin by comparing our predictions to two mRNA splicing datasets containing several cancer-susceptibility genes. Availability and implementation Source code is freely available under the Apache License, Version 2.0: https://github.com/Ensembl/VEP_plugins. Supplementary information Supplementary data are available at Bioinformatics online.

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