Sedimentation analysis of polyadenylation-specific complexes

1988 ◽  
Vol 8 (1) ◽  
pp. 226-233
Author(s):  
C L Moore ◽  
H Skolnik-David ◽  
P A Sharp
Keyword(s):  
Early Stage ◽  
Kinetic Studies ◽  
Nuclear Extract ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Processing ◽  
Precursor Rna ◽  
A Site ◽  
Nuclear Ribonucleoprotein ◽  
Sequence Requirements

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

scholarly journals Sedimentation analysis of polyadenylation-specific complexes.

1988 ◽  
Vol 8 (1) ◽  
pp. 226-233 ◽  
Author(s):  
C L Moore ◽  
H Skolnik-David ◽  
P A Sharp
Keyword(s):  
Early Stage ◽  
Kinetic Studies ◽  
Nuclear Extract ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Processing ◽  
Precursor Rna ◽  
A Site ◽  
Nuclear Ribonucleoprotein ◽  
Sequence Requirements

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

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

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.

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

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.

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

1987 ◽  
Vol 7 (1) ◽  
pp. 495-503 ◽  
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.

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

1987 ◽  
Vol 7 (1) ◽  
pp. 495-503
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.

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

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.

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

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.

scholarly journals A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

1995 ◽  
Vol 129 (4) ◽  
pp. 899-908 ◽  
Author(s):  
K M Neugebauer ◽  
J A Stolk ◽  
M B Roth
Keyword(s):  
Rna Polymerase Ii ◽  
Active Sites ◽  
Nuclear Extract ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Structural Element ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.

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.

scholarly journals In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

1990 ◽  
Vol 10 (8) ◽  
pp. 3868-3872 ◽  
Author(s):  
C M Shumard ◽  
C Torres ◽  
D C Eichler
Keyword(s):  
18S Rrna ◽  
Precise Determination ◽  
In Vivo Studies ◽  
Rrna Sequence ◽  
S1 Nuclease ◽  
In Vitro Processing ◽  
Rrna Maturation ◽  
A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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