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R Kole

Publications and source records attributed to R Kole.

At least 55 records · Page 3Linked to original sources
PubMed1992

A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins.

One of the consequences of the heat shock response is a shutdown of pre-mRNA splicing, a phenomenon that can be reproduced in extracts prepared from heat-shocked cells. The block in splicing occurs before the covalent modifications that generate spliced mRNA at the level of spliceosome formation. We have used extracts prepared from heat-shocked cells as a complementation system to characterize and partially purify a protein factor that is inactivated during the in vivo heat shock. The activity functions in the formation of the active spliceosome by assembling U4/U6 and U5 snRNPs into a triple snRNP particle. The factor appears to be different from previously isolated splicing factors and is functionally equivalent to several polypeptides that are specifically associated with the purified triple snRNP but not with individual U4/U6 or U5 snRNPs. Our data confirm the hypothesis that U4/U6 and U5 snRNPs enter the spliceosome as a triple snRNP complex and show for the first time a function of specific snRNP-associated polypeptides in the mammalian splicing pathway.

Blotting, Northern↗
PubMed1992

Cooperation of pre-mRNA sequence elements in splice site selection.

We have recently demonstrated that short internal exons in pre-mRNA transcripts with three exons and two introns are ignored by splicing machinery in vitro and in vivo, resulting in exon skipping. Exon skipping is reversed when the pyrimidine content of the polypyrimidine tract in the upstream intron is increased (Z. Dominski and R. Kole, Mol. Cell. Biol. 11:6075-6083, 1991). Here we show that skipping of the short internal exon can be partially reversed by mutations which modify the upstream branch point sequence of the 5' splice site at the end of the exon to their respective consensus sequences. When the modified elements are combined with one another in the same pre-mRNA, exon skipping is fully reversed. Full reversion of exon skipping is also observed when these elements are combined individually with the upstream polypyrimidine tract strengthened by three purine-to-pyrimidine mutations. The observed patterns of splice site selection are similar in vitro (in nuclear extracts from HeLa cells) and in vivo (in transfected HeLa cells). We also show that the length of the downstream intron plays a role in splice site selection. Our data indicate that the interplay between the sequence elements in pre-mRNA controls the outcome of each splicing event, providing the means for very subtle regulation of alternative splicing.

Base Composition↗
PubMed1991

Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera.

Antisense DNA oligodeoxynucleotides can selectively inhibit the expression of individual (undesirable) genes and thus, have potential in the treatment of cancer and viral diseases. A prerequisite to their use as therapeutic agents is information on the stability of oligodeoxynucleotides, and their structurally modified analogs, in the biological milieu. To this end, degradation of 5' end and internally [32P] labelled unmodified DNA oligodeoxynucleotide (D-oligo) and analogs containing phosphorothioate (S-oligo), methylphosphonate (MP-oligo), and novel alternating methylphosphonate and phosphodiester (Alt-MP-oligo) internucleoside linkages was studied in Hela cell nuclear extract, S100 cytoplasmic extract, normal human serum and calf serum at 37 degrees C. Both 5' end and internally labelled D-oligos showed complete degradation within 30 min incubation in human serum at 37 degrees C. In any given medium, the D-oligo was the least stable oligodeoxynucleotide to nuclease degradation whereas the Alt-MP, MP and S-oligos were generally of comparable stability and all relatively more stable than D-oligo. Interestingly, MP and Alt-MP-oligos also exhibited greater resistance to phosphatases in cellular extracts compared to D and S-oligos. Under the conditions of the experiments, increasing degradation for any given oligonucleotide was observed in the order: S100 cytoplasmic extract less than nuclear extract less than normal human serum less than calf serum. In a study involving alpha-MEM cell culture medium containing 10% heat inactivated fetal calf serum (heated to 56 degrees C for 1 hour), the D-oligo was found to be rapidly degraded (degradation evident within 10 mins) whereas degradation products for the S-oligo were observed within 1 hour. In contrast, the Alt-MP oligo remained stable throughout the 3 hour experiment. These results indicated that in cell culture medium containing heat inactivated serum Alt-MP oligo was more stable than D- and S-oligos.

Animals↗
PubMed1991

Selection of splice sites in pre-mRNAs with short internal exons.

Model pre-mRNAs containing two introns and three exons, derived from the human beta-globin gene, were used to study the effects of internal exon length on splice site selection. Splicing was assayed in vitro in HeLa nuclear extracts and in vivo during transient expression in transfected HeLa cells. For substrates with internal exons 87, 104, and 171 nucleotides in length, in vitro splicing proceeded via a regular splicing pathway, in which all three exons were included in the spliced product. Primary transcripts with internal exons containing 23, 29, and 33 nucleotides were spliced by an alternative pathway, in which the first exon was joined directly to the third one. The internal exon was missing from the spliced product and together with two flanking introns was included in a large lariat structure. The same patterns of splicing were retained when transcripts containing 171-, 33-, and 29-nucleotide-long internal exons were spliced in vivo. A transcript containing a 51-nucleotide-long exon was spliced in vitro via both pathways but in vivo generated only a correctly spliced product. Skipping of short internal exons was reversed both in vitro and in vivo when purines in the upstream polypyrimidine tract were replaced by pyrimidines. The changes in the polypyrimidine tract achieved by these substitutions led in vitro to complete (transcripts containing 28 pyrimidines in a row) or partial (transcripts containing 15 pyrimidines in a row) restoration of a regular splicing pathway. Splicing in vivo of these transcripts led exclusively to the spliced product containing all three exons. These results suggest that a balance between the length of the uninterrupted polypyrimidine tract and the length of the exon is an important determinant of the relative strength of the splice sites, ensuring correct splicing patterns of multiintron pre-mRNAs.

Base Sequence↗
PubMed1990

Inactivation of splicing factors in HeLa cells subjected to heat shock.

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.

Cell Nucleus↗
PubMed1989

RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds.

Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E. coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide. Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.

Base Sequence↗
PubMed1989

Inhibition of pre-mRNA splicing by 5-fluoro-, 5-chloro-, and 5-bromouridine.

Pre-mRNA transcripts of the human beta-globin gene containing 5-fluoro-, 5-chloro-, and 5-bromouridine were tested for splicing in vitro. Pre-mRNA containing 5-fluorouridine was spliced accurately and efficiently in the nuclear extract from HeLa cells, whereas 5-chloro-, and 5-bromouridine containing transcripts were not spliced. Analysis of the splicing reactions by electrophoresis on nondenaturing polyacrylamide gels showed that the latter two transcripts were unable to form active splicing complexes. Treatment of HeLa cell cultures with 5-fluorouridine decreased the splicing activity of the nuclear extracts in a dose- and time-dependent fashion. The decrease in splicing activity of these extracts appears to be due in part to a decreased level of U-2 small nuclear RNA and the corresponding ribonucleoprotein particle, U2-snRNP.

Antiviral Agents↗
PubMed1988

The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.

Base Sequence↗
PubMed1987

Alu RNA transcribed in vitro binds the 68-kDa subunit of the signal recognition particle.

Antibodies directed against the 68-kDa subunit of signal recognition particle (SRP) precipitate an Alu RNA X protein complex formed during in vitro transcription of a plasmid containing an Alu family sequence. The same Alu RNA X protein complex is precipitated by anti-Alu sera from certain patients with systemic lupus erythematosus and related autoimmune diseases (Kole, R., Fresco, L. D., Keene, J. D., Cohen, P. L., Eisenberg, R. A., and Andrews, P. G. (1985) J. Biol. Chem. 260, 11781-11786). Similarly to anti-SRP antibodies human, anti-Alu sera precipitate SRP from HeLa cell extract and detect a 68-kDa SRP subunit on immunoblots. These results indicate that the Alu antigen and the 68-kDa SRP subunit are the same polypeptide.

Animals↗
PubMed1987

RNA fingerprinting using a small horizontal agarose gel electrophoresis apparatus.

The electrophoresis step in the RNA fingerprinting procedure has been modified by replacing the large and expensive electrophoresis tank with a horizontal agarose gel electrophoresis box, a common piece of laboratory equipment. Two RNA molecules (67 and 298 nucleotides long) transcribed in vitro from a truncated human beta-globin gene have been fingerprinted and analyzed by the modified technique. The resolution of the oligonucleotides in this procedure is comparable to that seen using the tank and is sufficient to separate relatively large oligonucleotides with similar base composition.

Base Sequence↗
PubMed1986

Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA.

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing.

Antibody Specificity↗
PubMed1986

Inhibition of splicing but not cleavage at the 5' splice site by truncating human beta-globin pre-mRNA.

Human beta-globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. All of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps--i.e., cleavage at the 3' splice site and exon ligation--need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2+ are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.

Adenosine Triphosphate↗
PubMed1985

Alu RNA-protein complexes formed in vitro react with a novel lupus autoantibody.

We have screened sera from patients with systemic lupus erythematosus for reactivity with RNA transcribed in vitro using HeLa whole cell extracts. Sera from 14 out of 114 patients precipitated an RNA transcribed by RNA polymerase III from a plasmid containing an Alu family sequence (i.e. the repetitive DNA sequence that is cut by the Alu restriction enzyme) located upstream from the human gamma G-globin gene. These Alu transcripts were not precipitated by anti-La, anti-Sm, anti-RNP or anti-Ro antibodies, suggesting that Alu RNA was precipitated by a previously undescribed lupus specificity. Analysis of [35S]methionine-labeled immunoprecipitates indicated that Alu RNA binds a protein of about 68 kDa. This protein may be Alu specific since three different Alu transcripts were precipitated by the anti-Alu sera whereas another RNA polymerase III transcript, adenovirus VA I RNA, was not precipitated by the same sera.

Adenoviridae↗
PubMed1982

Accurate in vitro splicing of human beta-globin RNA.

Human beta-globin RNA transcribed from an exogenous DNA template is spliced in vitro by concentrated whole cell extracts from HeLa cells. Using the primer extension technique, we have shown that the small intervening sequence is spliced accurately and that the sequence of the product across the splice junction is identical to that of beta-globin mRNA prepared from human reticulocytes. The efficiency of the splicing reaction is low. The RNA transcript containing both introns and terminated upstream from the polyadenylation site is spliced most efficiently. The transcript which is terminated downstream from the polyadenylation site is not spliced at all. Thalassemic beta-globin RNA which carries an extra splice site in the small intron is also spliced, albeit with a low yield.

Base Sequence↗
PubMed1981

Properties of purified ribonuclease P from Escherichia coli.

The purified protein moiety of ribonuclease P (EC 3.1.26.5) from Escherichia coli, a single polypeptide of molecular weight approximately 17 500, has not catalytic activity by itself on several RNA substrates. However, when it is marked in vitro with an RNA species called M1 RNA, RNase P activity is reconstituted. The rate at which the purified RNase P cleaves any particular tRNA precursor molecule depends on the identity of that tRNA precursor.

Endonucleases↗
PubMed1980

E. coli RNAase P has a required RNA component.

RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the rnaase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro.

Escherichia coli↗
PubMed1979

Reconstitution of RNase P activity from inactive RNA and protein.

RNase P preparations from Escherichia coli can be separated into RNA and protein by chromatography, in buffers containing 7 M urea, on Sephadex G-200, DEAE-Sephadex, or CM-Sephadex columns. Neither RNA nor protein components alone exhibits any RNase activity. RNase P activity can be reconstituted by mixing separated RNA and protein components in buffer containing 7M urea followed by dialysis of this mixture to remove the urea. Of several purified RNAs tried, only M2 RNA, the RNA species found in purified RNase P, is active in the reconstitution experiments.

Escherichia coli↗