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

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.

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.

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.

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 U2 small nuclear RNA 3' end formation is directed by a critical internal structure distinct from the processing site.

1993 ◽  
Vol 13 (2) ◽  
pp. 1119-1129 ◽  
Author(s):  
M R Jacobson ◽  
M Rhoadhouse ◽  
T Pederson
Keyword(s):  
Rna Processing ◽  
Minor Effect ◽  
Small Nuclear Rna ◽  
Recognition Sequence ◽  
Stem Loop ◽  
Branch Site ◽  
Nuclear Rna ◽  
A Minor ◽  
Site Recognition

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.

scholarly journals A small nuclear RNA, U5, can transform cells in vitro.

1989 ◽  
Vol 9 (10) ◽  
pp. 4345-4356 ◽  
Author(s):  
K Hamada ◽  
T Kumazaki ◽  
K Mizuno ◽  
K Yokoro
Keyword(s):  
Cell Transformation ◽  
Rnase H ◽  
Low Molecular Weight ◽  
Small Nuclear Rna ◽  
Fibroblastic Cell ◽  
Myc Oncogene ◽  
Chemically Induced ◽  
Complementary Oligonucleotide ◽  
Nuclear Rna

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.

scholarly journals U1 small nuclear ribonucleoprotein studied by in vitro assembly.

1983 ◽  
Vol 96 (6) ◽  
pp. 1751-1755 ◽  
Author(s):  
E D Wieben ◽  
S J Madore ◽  
T Pederson
Keyword(s):  
Direct Analysis ◽  
Small Nuclear Rna ◽  
In Vitro System ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Specific Complex ◽  
Reticulocyte Lysate

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

scholarly journals The Xenopus laevis U2 gene distal sequence element (enhancer) is composed of four subdomains that can act independently and are partly functionally redundant.

1989 ◽  
Vol 9 (4) ◽  
pp. 1682-1690 ◽  
Author(s):  
G Tebb ◽  
I W Mattaj
Keyword(s):  
Transcription Factor ◽  
Xenopus Oocytes ◽  
Small Nuclear Rna ◽  
Base Pairs ◽  
Sequence Element ◽  
Nuclear Factors ◽  
Nuclear Rna ◽  
The Individual ◽  
Mutant Genes

The sequences involved in enhancement of transcription of the Xenopus U2 small nuclear RNA gene by the distal sequence element (DSE) of its promoter were analyzed in detail by microinjection of mutant genes into Xenopus oocytes. The DSE was shown to be roughly 60 base pairs long. Within this region, four motifs were found to contribute to DSE function: an ATGCAAAT octamer sequence, an SpI binding site, and two additional motifs which, since they are related in sequence, may bind the same transcription factor. These motifs were named D2 (for DSE; U2). Both the octamer sequence and the SpI site bound nuclear factors in vitro, but no factor binding to the D2 motifs was detected. All four elements were independently capable of enhancing transcription of the U2 gene to some extent. Furthermore, when assayed under both competitive and noncompetitive conditions, the individual units of the DSE displayed functional redundancy.

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 Transient Nucleolar Localization Of U6 Small Nuclear RNA InXenopus Laevis Oocytes

2000 ◽  
Vol 11 (7) ◽  
pp. 2419-2428 ◽  
Author(s):  
Thilo Sascha Lange ◽  
Susan A. Gerbi
Keyword(s):  
Nucleolar Localization ◽  
Xenopus Laevis Oocytes ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Nuclear Rna ◽  
Inxenopus Laevis ◽  
Nucleolar Staining ◽  
Over Time

Recent studies on the 2′-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

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