Human genes for U2 small nuclear RNA are tandemly repeated

1984 ◽  
Vol 4 (3) ◽  
pp. 492-499
Author(s):  
S W Van Arsdell ◽  
A M Weiner
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Initiation Site ◽  
Tandem Array ◽  
Small Nuclear Rna ◽  
Coding Region ◽  
Region I ◽  
Nuclear Rna ◽  
Region Ii

We found that the genes for human U2 small nuclear RNA (snRNA) are organized as a nearly perfect tandem array of 10 to 20 copies per haploid genome. Although the coding region for the mature form of U2 RNA was only 188 base pairs (bp) long, the basic repeating unit of the tandem array was 6 kilobase pairs in length. Comparison of DNA sequences immediately upstream from human U1 and U2 genes revealed two regions of strong homology: region I (15 bp long) lay upstream of region II (20 bp long) and was separated from it by about the same distance in U1 genes (25 bp) as in U2 genes (21 bp); however, region I and region II were located 174 bp further upstream from the 5' end of the snRNA coding sequence in U1 genes than in U2 genes. Homologs of region II were also found upstream of the snRNA coding region in a mouse U2 gene and two rat U1 genes. Murphy et al. (Cell 29:265-274, 1982) have found that sequences within region II may function as the equivalent of a TATA box for initiation by RNA polymerase II in vitro at a position 183 bp upstream from the 5' end of the human U1 snRNA coding region. In light of the data reported here, this result suggests that region II does indeed play a role in transcription but that its position relative to the actual initiation site can vary.

scholarly journals Human genes for U2 small nuclear RNA are tandemly repeated.

1984 ◽  
Vol 4 (3) ◽  
pp. 492-499 ◽  
Author(s):  
S W Van Arsdell ◽  
A M Weiner
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Initiation Site ◽  
Tandem Array ◽  
Small Nuclear Rna ◽  
Coding Region ◽  
Region I ◽  
Nuclear Rna ◽  
Region Ii

We found that the genes for human U2 small nuclear RNA (snRNA) are organized as a nearly perfect tandem array of 10 to 20 copies per haploid genome. Although the coding region for the mature form of U2 RNA was only 188 base pairs (bp) long, the basic repeating unit of the tandem array was 6 kilobase pairs in length. Comparison of DNA sequences immediately upstream from human U1 and U2 genes revealed two regions of strong homology: region I (15 bp long) lay upstream of region II (20 bp long) and was separated from it by about the same distance in U1 genes (25 bp) as in U2 genes (21 bp); however, region I and region II were located 174 bp further upstream from the 5' end of the snRNA coding sequence in U1 genes than in U2 genes. Homologs of region II were also found upstream of the snRNA coding region in a mouse U2 gene and two rat U1 genes. Murphy et al. (Cell 29:265-274, 1982) have found that sequences within region II may function as the equivalent of a TATA box for initiation by RNA polymerase II in vitro at a position 183 bp upstream from the 5' end of the human U1 snRNA coding region. In light of the data reported here, this result suggests that region II does indeed play a role in transcription but that its position relative to the actual initiation site can vary.

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

scholarly journals In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927 ◽  
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

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.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

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 In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

Multiple functional motifs in the chicken U1 RNA gene enhancer

1987 ◽  
Vol 7 (12) ◽  
pp. 4185-4193
Author(s):  
K A Roebuck ◽  
R J Walker ◽  
W E Stumph
Keyword(s):  
Gene Expression ◽  
Dna Sequences ◽  
Initiation Site ◽  
Sequence Motifs ◽  
Small Nuclear Rna ◽  
Multiple Sequence ◽  
Transcriptional Initiation ◽  
Transcription System ◽  
Cell Extracts ◽  
Gene Enhancer

The DNA sequence requirements of chicken U1 RNA gene expression have been examined in an oocyte transcription system. An enhancer region, which was required for efficient U1 RNA gene expression, is contained within a region of conserved DNA sequences spanning nucleotide positions -230 to -183, upstream of the transcriptional initiation site. These DNA sequences can be divided into at least two distinct subregions or domains that acted synergistically to provide a greater than 20-fold stimulation of U1 RNA synthesis. The first domain contains the octamer sequence ATGCAAAT and was recognized by a DNA-binding factor present in HeLa cell extracts. The second domain (the SPH domain) consists of conserved sequences immediately downstream of the octamer and is an essential component of the enhancer. In the oocyte, the DNA sequences of the SPH domain were able to enhance gene expression at least 10-fold in the absence of the octamer domain. In contrast, the octamer domain, although required for full U1 RNA gene activity, was unable to stimulate expression in the absence of the adjacent downstream DNA sequences. These findings imply that sequences 3' of the octamer play a major role in the function of the chicken U1 RNA gene enhancer. This concept was supported by transcriptional competition studies in which a cloned chicken U4B RNA gene was used to compete for limiting transcription factors in oocytes. Multiple sequence motifs that can function in a variety of cis-linked configurations may be a general feature of vertebrate small nuclear RNA gene enhancers.

Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract

1987 ◽  
Vol 7 (9) ◽  
pp. 3131-3137
Author(s):  
A M Kleinschmidt ◽  
T Pederson
Keyword(s):  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Rnase Protection ◽  
In Vitro Processing ◽  
S100 Extract ◽  
Processing Activity ◽  
Precursor Molecules ◽  
Trna Precursor ◽  
Rna Precursor

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

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