A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

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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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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.12.4.1460-1468.1992 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1460-1468

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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PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3939 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3939-3947 ◽

Cited By ~ 41

Author(s):

S Blanton ◽

A Srinivasan ◽

B C Rymond

Keyword(s):

Structural Similarity ◽

Mrna Splicing ◽

Heat Inactivation ◽

Temperature Sensitive ◽

Small Nuclear Rna ◽

Virtual Absence ◽

U6 Snrna ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Rna

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.

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PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3939-3947.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3939-3947

Author(s):

S Blanton ◽

A Srinivasan ◽

B C Rymond

Keyword(s):

Structural Similarity ◽

Mrna Splicing ◽

Heat Inactivation ◽

Temperature Sensitive ◽

Small Nuclear Rna ◽

Virtual Absence ◽

U6 Snrna ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Rna

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.

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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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The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5377-5382.1993 ◽

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.

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Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4479-4487.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4479-4487

Author(s):

M Cotten ◽

G Schaffner ◽

M L Birnstiel

Keyword(s):

Antisense Rna ◽

Nuclear Extract ◽

Mrna Processing ◽

Rna Molecules ◽

Small Nuclear Ribonucleoprotein ◽

In Vitro Processing ◽

Antisense Dna ◽

Nuclear Ribonucleoprotein ◽

Fold Excess

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

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A novel protein factor is required for use of distal alternative 5' splice sites in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5945-5953.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5945-5953

Author(s):

J E Harper ◽

J L Manley

Keyword(s):

Splice Site ◽

Site Selection ◽

Deae Cellulose ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Splice Sites ◽

Splice Site Selection ◽

Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

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Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.495 ◽

1987 ◽

Vol 7 (1) ◽

pp. 495-503 ◽

Cited By ~ 18

Author(s):

L C Ryner ◽

J L Manley

Keyword(s):

Simian Virus 40 ◽

Nuclear Extract ◽

Structural Features ◽

Simian Virus ◽

Micrococcal Nuclease ◽

Small Nuclear Ribonucleoprotein ◽

Cleavage And Polyadenylation ◽

Nuclear Ribonucleoprotein ◽

Cell Nuclear Extract

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

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