scholarly journals Architecture of the U5 small nuclear RNA.

1994 ◽  
Vol 14 (3) ◽  
pp. 2180-2190 ◽  
Author(s):  
D N Frank ◽  
H Roiha ◽  
C Guthrie
Keyword(s):  
Secondary Structure ◽  
Comparative Sequence Analysis ◽  
Fungal Species ◽  
Deletion Analysis ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Internal Loop ◽  
Nuclear Rna ◽  
Sequence Elements ◽  
U5 Snrna

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

Architecture of the U5 small nuclear RNA

1994 ◽  
Vol 14 (3) ◽  
pp. 2180-2190
Author(s):  
D N Frank ◽  
H Roiha ◽  
C Guthrie
Keyword(s):  
Secondary Structure ◽  
Comparative Sequence Analysis ◽  
Fungal Species ◽  
Deletion Analysis ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Internal Loop ◽  
Nuclear Rna ◽  
Sequence Elements ◽  
U5 Snrna

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

Secondary structure of U6 small nuclear RNA: implications for spliceosome assembly

2010 ◽  
Vol 38 (4) ◽  
pp. 1099-1104 ◽  
Author(s):  
Elizabeth A. Dunn ◽  
Stephen D. Rader
Keyword(s):  
Secondary Structure ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
New Model ◽  
Rna Molecules ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
High Level ◽  
And Function

U6 snRNA (small nuclear RNA), one of five RNA molecules that are required for the essential process of pre-mRNA splicing, is notable for its high level of sequence conservation and the important role it is thought to play in the splicing reaction. Nevertheless, the secondary structure of U6 in the free snRNP (small nuclear ribonucleoprotein) form has remained elusive, with predictions changing substantially over the years. In the present review we discuss the evidence for existing models and critically evaluate a fundamental assumption of these models, namely whether the important 3′ ISL (3′ internal stem–loop) is present in the free U6 particle, as well as in the active splicing complex. We compare existing models of free U6 with a newly proposed model lacking the 3′ ISL and evaluate the implications of the new model for the structure and function of U6's base-pairing partner U4 snRNA. Intriguingly, the new model predicts a role for U4 that was unanticipated previously, namely as an activator of U6 for assembly into the splicing machinery.

scholarly journals Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2782-2790 ◽  
Author(s):  
Véronique Ségault ◽  
Cindy L. Will ◽  
Maria Polycarpou-Schwarz ◽  
Iain W. Mattaj ◽  
Christiane Branlant ◽  
...  
Keyword(s):  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Internal Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna ◽  
Loop 2

ABSTRACT The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa orXenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5′ and 3′ halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

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 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 Sensitivity to UV radiation of small nuclear RNA synthesis in mammalian cells.

1983 ◽  
Vol 3 (12) ◽  
pp. 2151-2155 ◽  
Author(s):  
G L Eliceiri ◽  
J H Smith
Keyword(s):  
Uv Radiation ◽  
Uv Irradiation ◽  
Mammalian Cells ◽  
Repair Process ◽  
Rrna Synthesis ◽  
Small Nuclear Rna ◽  
Pyrimidine Dimers ◽  
5.8S Rrna ◽  
Nuclear Rna ◽  
U5 Snrna

It was demonstrated previously that the synthesis of small nuclear RNA (snRNA) species U1 and U2 in human cells is very sensitive to UV radiation. In the present work, the UV sensitivity of U3, U4, and U5 snRNA synthesis is shown to be also high. The synthesis of U1, U2, U3, U4, and U5 snRNAs progressively decreased during the first 2 h after UV irradiation (this was not observed in polyadenylated RNA) and had not returned to normal rates 6 h after UV exposure. In contrast, the restoration of 5.8S rRNA synthesis began immediately after UV irradiation and was essentially complete 6 h later. A small fraction of U1 and U5 (and possibly U2 and U3) snRNA synthesis remained unaffected by high UV doses, when cell radiolabeling began 10 min after UV irradiation. The present data suggest that a factor other than the level of pyrimidine dimers in DNA (possibly, steps in the post-irradiation DNA repair process) plays an important role in the mechanism of UV-induced inhibition of U1-U5 snRNA synthesis.

scholarly journals Structural and functional analysis of chicken U4 small nuclear RNA genes.

1986 ◽  
Vol 6 (11) ◽  
pp. 3910-3919 ◽  
Author(s):  
M L Hoffman ◽  
G M Korf ◽  
K J McNamara ◽  
W E Stumph
Keyword(s):  
Distal Region ◽  
Xenopus Laevis Oocytes ◽  
Small Nuclear Rna ◽  
Transcriptional Enhancer ◽  
Base Pairs ◽  
Base Substitutions ◽  
Nuclear Rna ◽  
Sequence Elements ◽  
U4 Rna ◽  
Rna Genes

Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion.

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.

scholarly journals Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

1991 ◽  
Vol 11 (7) ◽  
pp. 3432-3445 ◽  
Author(s):  
D A Wassarman ◽  
J A Steitz
Keyword(s):  
Secondary Structure ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Base Pairing ◽  
Structural Determinants ◽  
Nuclear Rna ◽  
Sequence Complementarity ◽  
Associated Proteins ◽  
Stem Structure

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.

scholarly journals Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids

1999 ◽  
Vol 19 (2) ◽  
pp. 1595-1604 ◽  
Author(s):  
Nancy R. Sturm ◽  
Michael C. Yu ◽  
David A. Campbell
Keyword(s):  
Binding Site ◽  
Transcription Termination ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Spliced Leader ◽  
Nuclear Extracts ◽  
Sequence Elements ◽  
Rna Genes

ABSTRACT Addition of a 39-nucleotide (nt) spliced leader (SL) bytrans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3′ end of all kinetoplastid SL RNA genes, and that more than six T’s are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3′ ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T’s. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3′ end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3′-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

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