The U1 small nuclear RNA-protein complex selectively binds a 5′ splice site in vitro

Cell ◽  
1983 ◽  
Vol 33 (2) ◽  
pp. 509-518 ◽  
Author(s):  
Stephen M. Mount ◽  
Ingvar Pettersson ◽  
Monique Hinterberger ◽  
Aavo Karmas ◽  
Joan A. Steitz
Keyword(s):  
Splice Site ◽  
Protein Complex ◽  
Small Nuclear Rna ◽  
Nuclear Rna

scholarly journals A TFIIH-Associated Mediator Head Is a Basal Factor of Small Nuclear Spliced Leader RNA Gene Transcription in Early-Diverged Trypanosomes

2010 ◽  
Vol 30 (23) ◽  
pp. 5502-5513 ◽  
Author(s):  
Ju Huck Lee ◽  
Gang Cai ◽  
Aswini K. Panigrahi ◽  
Star Dunham-Ems ◽  
Tu N. Nguyen ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Protein Complex ◽  
Complex Form ◽  
Amino Acid Sequences ◽  
Small Nuclear Rna ◽  
Basal Transcription ◽  
Spliced Leader ◽  
Nuclear Rna

ABSTRACT Genome annotation suggested that early-diverged kinetoplastids possess a reduced set of basal transcription factors. More recent work, however, on the lethal parasite Trypanosoma brucei identified extremely divergent orthologs of TBP, TFIIA, TFIIB, and TFIIH which, together with the small nuclear RNA-activating protein complex, form a transcription preinitiation complex (PIC) at the spliced leader (SL) RNA gene (SLRNA) promoter. The SL RNA is a small nuclear RNA and a trans splicing substrate for the maturation of all pre-mRNAs which is metabolized continuously to sustain gene expression. Here, we identified and biochemically characterized a novel TFIIH-associated protein complex in T. brucei (Med-T) consisting of nine subunits whose amino acid sequences are conserved only among kinetoplastid organisms. Functional analyses in vivo and in vitro demonstrated that the complex is essential for cell viability, SLRNA transcription, and PIC integrity. Molecular structure analysis of purified Med-T and Med-T/TFIIH complexes by electron microscopy revealed that Med-T corresponds to the mediator head module of higher eukaryotes. These data therefore show that mediator is a basal factor for small nuclear SL RNA gene transcription in trypanosomes and that the basal transcription function of mediator head is a characteristic feature of eukaryotes which developed early in their evolution.

scholarly journals Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (9) ◽  
pp. 6337-6349 ◽  
Author(s):  
S E Wells ◽  
M Ares
Keyword(s):  
Synthetic Lethality ◽  
Small Nuclear Rna ◽  
Rna Structures ◽  
Stem Loop ◽  
U2 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

U2.3 Precursor Small Nuclear RNA in vitro Processing Assay

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (17) ◽  
Author(s):  
Chan Lin ◽  
Yujie Feng ◽  
Xueyan Peng ◽  
Jiaming Wu ◽  
Weili Wang ◽  
...  
Keyword(s):  
Small Nuclear Rna ◽  
In Vitro Processing ◽  
Nuclear Rna

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

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.

Crystal structure of the spliceosomal U2B″–U2A′ protein complex bound to a fragment of U2 small nuclear RNA

Nature ◽  
10.1038/29234 ◽  
1998 ◽  
Vol 394 (6694) ◽  
pp. 645-650 ◽  
Author(s):  
Stephen R. Price ◽  
Philip R. Evans ◽  
Kiyoshi Nagai
Keyword(s):  
Crystal Structure ◽  
Protein Complex ◽  
Small Nuclear Rna ◽  
Nuclear Rna

scholarly journals Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

1990 ◽  
Vol 10 (3) ◽  
pp. 939-946 ◽  
Author(s):  
R Singh ◽  
S Gupta ◽  
R Reddy
Keyword(s):  
Cell Extract ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Rna Sequence ◽  
U6 Snrna ◽  
Stem Loop Structure ◽  
Close Proximity ◽  
Nuclear Rna ◽  
Tightly Coupled

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

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