Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem

1992 ◽  
Vol 12 (10) ◽  
pp. 4456-4463
Author(s):  
G M Huang ◽  
A Jarmolowski ◽  
J C Struck ◽  
M J Fournier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
Essential Elements ◽  
Structural Elements ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Compensatory Base Changes ◽  
Normal Processing ◽  
Intragenic Suppressor

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.

scholarly journals Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem.

1992 ◽  
Vol 12 (10) ◽  
pp. 4456-4463 ◽  
Author(s):  
G M Huang ◽  
A Jarmolowski ◽  
J C Struck ◽  
M J Fournier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
Essential Elements ◽  
Structural Elements ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Compensatory Base Changes ◽  
Normal Processing ◽  
Intragenic Suppressor

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.

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.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577 ◽  
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

U2 small nuclear RNA is remarkably conserved between Schizosaccharomyces pombe and mammals

1988 ◽  
Vol 8 (12) ◽  
pp. 5575-5580
Author(s):  
P Brennwald ◽  
G Porter ◽  
J A Wise
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secondary Structure ◽  
Schizosaccharomyces Pombe ◽  
Small Nuclear Rna ◽  
Primary Sequence ◽  
Cloning And Sequencing ◽  
Sequence Identity ◽  
Human Counterpart ◽  
Nuclear Rna ◽  
Rna Blot Analysis

We report the molecular cloning and sequencing of the most abundant trimethylguanosine-capped small nuclear RNA from the fission yeast Schizosaccharomyces pombe, a highly conserved homolog of mammalian U2 small nuclear RNA. This RNA is 186 nucleotides in length, just 2 nucleotides shorter than its human counterpart; this is in contrast to Saccharomyces cerevisiae U2, which is 1,175 nucleotides long. Moreover, the secondary structure of Schizosaccharomyces pombe U2 is virtually identical to that of mammalian U2, including the 3' half of the RNA, which shows limited primary sequence identity. Northern (RNA) blot analysis revealed that the size of this RNA is conserved not only in fission yeasts but in many organisms, including other ascomycetes.

scholarly journals Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA in Saccharomyces cerevisiae

Genetics ◽  
2009 ◽  
Vol 184 (1) ◽  
pp. 107-118 ◽  
Author(s):  
Jonathan S. Finkel ◽  
Karen Chinchilla ◽  
Doris Ursic ◽  
Michael R. Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Nuclear Rna ◽  
Separable Functions ◽  
Nuclear Rna

scholarly journals Splicing of the Meiosis-Specific HOP2Transcript Utilizes a Unique 5′ Splice Site

1999 ◽  
Vol 19 (12) ◽  
pp. 7933-7943 ◽  
Author(s):  
Jun-Yi Leu ◽  
G. Shirleen Roeder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Splice Site ◽  
Meiotic Prophase ◽  
Nucleotide Substitution ◽  
Small Nuclear Rna ◽  
Single Nucleotide Substitution ◽  
Coding Region ◽  
Single Nucleotide ◽  
Nuclear Rna ◽  
Splicing Efficiency

ABSTRACT The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis. The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase. The HOP2 coding region is interrupted by an intron located near the 5′ end of the gene. This intron contains a nonconsensus 5′ splice site (GUUAAGU) that differs from the consensus 5′ splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution. Bases flanking the HOP2 5′ splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency. HOP2pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

scholarly journals Developmental and tissue-specific expression of U4 small nuclear RNA genes.

1988 ◽  
Vol 8 (12) ◽  
pp. 5566-5569 ◽  
Author(s):  
G M Korf ◽  
I W Botros ◽  
W E Stumph
Keyword(s):  
Domestic Chicken ◽  
Sequence Variant ◽  
Small Nuclear Rna ◽  
Specific Expression ◽  
Small Nuclear Rnas ◽  
Specific Manner ◽  
Nuclear Rna ◽  
U4 Rna ◽  
Rna Genes

U4 RNA is one of several small nuclear RNAs involved in the splicing of mRNA precursors. The domestic chicken has two genes per haploid genome that are capable of encoding U4 RNA. The U4X RNA gene (which encodes a sequence variant of U4 RNA that was unknown prior to the cloning of the gene) and the U4B RNA gene were both expressed in vivo in each of seven adult and three embryonic chicken tissues examined. However, the ratio of U4B RNA to U4X RNA can vary more than sevenfold in both a tissue- and stage-specific manner.

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