Early commitment of yeast pre-mRNA to the spliceosome pathway.

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6337 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6337-6349 ◽

Cited By ~ 27

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.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6337-6349.1994 ◽

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.

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Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5516-5526.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5516-5526 ◽

Cited By ~ 2

Author(s):

M Cross ◽

A Günzl ◽

Z Palfi ◽

A Bindereif

Keyword(s):

Trypanosoma Brucei ◽

Core Protein ◽

Rnase H ◽

Spliced Leader ◽

The Core ◽

In Vitro Reconstitution ◽

U2 Snrnp ◽

Protein Components ◽

Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

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N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4456-4465.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4456-4465

Author(s):

S M Carroll ◽

P Narayan ◽

F M Rottman

Keyword(s):

Consensus Sequence ◽

Specific Sequence ◽

Free System ◽

Consensus Sequences ◽

Neplanocin A ◽

A Cell ◽

Steady State Levels ◽

Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

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Tec1p-Independent Activation of a Hypha-Associated Candida albicans Virulence Gene during Infection

Infection and Immunity ◽

10.1128/iai.72.4.2386-2389.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2386-2389 ◽

Cited By ~ 6

Author(s):

Peter Staib ◽

Ayfer Binder ◽

Marianne Kretschmar ◽

Thomas Nichterlein ◽

Klaus Schröppel ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Candida Albicans ◽

Animal Models ◽

Environmental Conditions ◽

Deletion Mutant ◽

Virulence Gene ◽

Consensus Sequences ◽

Site Consensus

ABSTRACT The Tec1p transcription factor is involved in the expression of hypha-specific genes in Candida albicans. Although the induction of the hypha-associated SAP5 gene by serum in vitro depends on Tec1p, deletion of all Tec1p binding site consensus sequences from the SAP5 promoter did not affect its activation. In two different animal models of candidiasis, the SAP5 promoter was induced even in a Δtec1 deletion mutant, demonstrating that the requirement for Tec1p in gene expression in C. albicans depends on the environmental conditions within the host.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4345 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4345-4356 ◽

Cited By ~ 17

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.

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N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4456 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4456-4465 ◽

Cited By ~ 63

Author(s):

S M Carroll ◽

P Narayan ◽

F M Rottman

Keyword(s):

Consensus Sequence ◽

Specific Sequence ◽

Free System ◽

Consensus Sequences ◽

Neplanocin A ◽

A Cell ◽

Steady State Levels ◽

Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

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Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1460 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1460-1468 ◽

Cited By ~ 17

Author(s):

C Wersig ◽

A Bindereif

Keyword(s):

Mutational Analysis ◽

Nuclear Extract ◽

Small Nuclear Rna ◽

Interaction Domain ◽

Sequence Element ◽

Spliceosome Assembly ◽

Small Nuclear Ribonucleoprotein ◽

U4 Rna ◽

Nuclear Ribonucleoprotein

We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.

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Transient Nucleolar Localization Of U6 Small Nuclear RNA InXenopus Laevis Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2419 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2419-2428 ◽

Cited By ~ 40

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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