Biomedical subjects
C J Goldenberg
Publications and source records attributed to C J Goldenberg.
Purified human factor activates heat shock promoter in a HeLa cell-free transcription system.
Heat shock protein (hsp) genes are typically silent and are activated by various stresses including heat. As a first step toward understanding this activation event, a human factor, referred to here as human heat shock transcription factor (human HTF), has been purified approximately 14,000-fold from extracts of heat-treated HeLa cells by means of sequence-specific DNA affinity chromatography. The most highly purified fraction of human HTF binds specifically to the known regulatory sequence element (HSE) of hsp genes as shown by footprinting experiments. Purified human HTF has an apparent molecular mass of 83 kDa. Human HTF is specifically required for activation of an hsp gene promoter in a reconstituted in vitro transcription system from human cells. Activation is dependent on the presence of the HSEs in the transcription template.
Virus-specific splicing inhibitor in extracts from cells infected with HIV-1.
Human immunodeficiency virus type 1 (HIV-1), in contrast with most other retroviruses, encodes trans-regulatory proteins for virus gene expression. It is shown in this study, by means of an in vitro splicing system, that nuclear extracts obtained from cells infected with HIV-1 contain a factor (or factors) that specifically inhibits splicing of a synthetic SP6/HIV pre-messenger RNA (pre-mRNA)-containing donor and acceptor splice sites in the coding region for the envelope protein. It is also shown that the SP6/HIV pre-mRNA is not capable of assembly in a ribonucleoprotein complex, spliceosome, in extracts from infected cells. These findings raise the possibility that specific inhibition of pre-mRNA splicing in the envelope protein coding region by HIV-1 trans-regulatory factors might be one control mechanism for efficient production of structural viral proteins and virion assembly.
Intron sequences and the length of the downstream second exon affect the binding of hnRNP C proteins in an in vitro splicing reaction.
The proteins that are in direct contact with the pre-mRNA in an in vitro splicing reaction were analyzed by UV cross-linking experiments. Six major proteins (120, 55, 44, 42, 39 and 38 KD) and three minor polypeptides (84, 72 and 63 KD) were detected. The predominant proteins 44, 42 KD belong to the class of hnRNP C proteins since they were immunoprecipitated by monoclonal antibodies directed against hnRNP C proteins. The cross-linked proteins were not detected in the absence of Mg2+, ATP or when RNA lacking introns were used as substrates in the splicing reactions. The effect of exon sequences on the binding efficiency for the photocrosslinked proteins was investigated. Transcripts containing a second exon of 24 nucleotides for the beta-globin or 107 nucleotides for the mouse insulin, yielded a reduced amount of cross-linked proteins when compared with "full length" pre-mRNAs. Sequences within the first exon of the beta-globin pre-mRNA did not affect the binding efficiency of these proteins. The reduced binding efficiency of the cross-linked proteins for the truncated beta-globin or mouse insulin pre-mRNAs correlated with the lower efficiency for in vitro splicing. Substitutions with unrelated sequences in the beta-globin second exon restore the binding of the cross-linked proteins indicating that the length of the second exon and not specific sequences are relevant for the binding efficiency of these proteins. The SP6/mouse insulin oligonucleotides cross-linked to the hnRNP C proteins were isolated and sequenced. A 17-mer was located in the second exon (134 nucleotides downstream from the 3' splice site) and a 14-mer in the intron region (25 nucleotides downstream the 5' splice site). The beta-globin oligonucleotides cross-linked to the hnRNP C proteins were a 13-mer in the second exon (28 nucleotides downstream the 3' splice site) and an 8-mer in the first exon (81 nucleotides downstream the 5' end of the pre-mRNA). Our results indicate that the hnRNP C proteins interact with those oligonucleotides located in different regions of the pre-mRNA. The binding efficiency of those proteins, however, depends on the length of the second exon and the presence of intron sequences (secondary and/or tertiary pre-mRNA structure).
The secondary structure of a messenger RNA precursor probed with psoralen is melted in an in vitro splicing reaction.
The secondary structure of the SP6/mouse insulin precursor RNA was determined by psoralen cross-linking experiments. A series of long-range contacts occur within the left half of the pre-mRNA that contains the intervening sequence. Multiple secondary structures for the pre-mRNA exist since some of the interactions share common sites. In splicing buffer but without the splicing extract added, many of these interactions are stable up to at least 50 degrees C. These interactions, however, are dissociated during the in vitro splicing reaction. This dissociation requires ATP and it occurs during the first 30 min. of the splicing reaction. Pre-mRNAs containing psoralen cross-links in different locations within the RNA molecule were purified and used as substrates for in vitro splicing. Psoralen cross-links at any of the double-stranded regions resulted in complete inhibition of the splicing reaction. This indicates that destabilization of the secondary structure of the SP6/mouse insulin pre-mRNA is necessary for in vitro splicing.
Inhibition of in vitro splicing of a mouse insulin pre-mRNA by covalent cross-links in the intron region.
Recent studies have indicated that in vitro splicing of a mouse insulin pre-mRNA by a HeLa cell nuclear extract is accompanied by the unwinding of substrate RNA. The present experiments were performed to determine whether this melting of the secondary structure of the precursor RNA is essential for the splicing reaction. 32P-labelled mouse insulin pre-mRNA synthesized in vitro in a SP6 transcription system was cross-linked with aminomethyltrimethyl psoralen and fractionated by polyacrylamide gel electrophoresis. RNA species containing different intramolecular cross-links were eluted from the gel and the sites of cross-links were mapped by primer extension analysis using synthetic oligonucleotide primers. Under conditions that allow accurate in vitro splicing of intact pre-mRNA, precursor molecules with psoralen cross-links within their intron region were not spliced by a HeLa cell nuclear extract. This observation strongly supports the assumption that unwinding of precursor RNA molecules is necessary for the splicing reaction.
Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex.
An SP6/mouse insulin RNA precursor containing two exons and one intron can be spliced in a partially purified nuclear extract isolated from MOPC-315 mouse myeloma cells. We have detected the putative RNA splicing intermediate (intron-3'exon) in a lariat form, the excised intron in a lariat form, and the mRNA spliced product. The in vitro splicing reaction of gel-purified RNA precursors requires ATP and Mg2+ and was accompanied by the formation of a 60-40S ribonucleoprotein complex. The formation of the 60S complex requires ATP. At least two Sm snRNPs containing U1 and U2 RNAs are components of the 60-40S complex. The assemble of those snRNPs occurs early during the splicing reaction and it requires ATP and intron containing pre-mRNAs.
Partial purification and properties of a pre-mRNA splicing activity.
Precursor RNA substrates for splicing reaction were synthesized in vitro from a plasmid DNA in which the early region 2 gene of adenovirus 2 was fused to an efficient bacteriophage promoter (Salmonella phage 6). Pre-mRNA splicing activity from nuclear extracts of MOPC-315 mouse myeloma cells was partially purified 108-fold by three chromatographic steps. The in vitro splicing reaction catalyzed by the partially purified fractions was efficient (60-80% substrate conversion) and accurate at the nucleotide level. The reaction occurred with crude or purified fractions without any detectable lag and nucleotides (ATP or GTP) were absolutely required. Monoclonal anti-Sm antibodies that quantitatively immunoprecipitate U1 small nuclear ribonucleoprotein particles totally inhibited the splicing activity of the purified fractions, indicating that U1 small nuclear RNPs had co-purified with the activity and were absolutely required for the splicing reaction.
Characterization of the newly-formed internucleotide bond of in vitro spliced mRNAs.
Precursor RNAs were synthesized in vitro from a plasmid in which the early region 2 (E2) of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and utilized as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. We have shown previously (Goldenberg, C.J., PNAS, August, in press, 1984) that in vitro splicing in those extracts was accurate at the nucleotide level. We now show that: i) the new internucleotide bond at the splice junction generated in vitro is a 3',5'-phosphodiester bond; and ii) the phosphate that forms the splice between the exons is derived from the pre-mRNA.
In vitro-synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts.
Precursor mRNAs were synthesized in vitro from a plasmid in which the early region 2 gene of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and used as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro splicing was accurate at the nucleotide level. The reaction occurs rapidly and without any detectable lag. The concentration of the pre-mRNA precursor during incubation appears to be an important factor for high efficiency (60%-80%) of in vitro RNA splicing. Fractionation of the splicing components as well as modifications of the DNA template to study the nucleotide-sequence requirement for in vitro splicing can now be accomplished with this system.
Accurate and efficient in vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.
Polyadenylated and deproteinized nuclear RNA precursors encoded by early region 2 of the adenovirus 2 genome are spliced in vitro by nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The nucleotide sequence across the splice junctions in the E2 RNAs processed in vitro was investigated by performing primer extensions in the presence of dideoxynucleotides and direct sequencing on polyacrylamide gels. We conclude that the in vitro splicing reaction is accurate and has the same precision as that of in vivo E2 cytoplasmic mRNA prepared from Ad2 infected cells. The efficiency of in vitro splicing by the nuclear extracts is very high. Approximately 80% of E2 RNA precursor, on a molar basis, are spliced in vitro to a mature RNA. These findings provide evidence that a nuclear extract prepared from MOPC-315 mouse myeloma cells is capable of accurate and efficient splicing of E2 RNA precursors.
In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.
Early region 2 of the adenovirus 2 genome (map position 61-75) specifies two poly(A)+ nuclear RNAs (28S and 23S) that appear to be precursors of the 20S cytoplasmic mRNA [Goldenberg, C. J. & Raskas, H. J. (1979) Cell 16, 131-138]. Splicing of these nuclear RNAs in vitro has been obtained with a whole cell extract prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The splicing reaction was demonstrated by two procedures: (i) hybridization of pulse-labeled RNA fractionated by size and (ii) annealing of RNAs with radioactive DNA probes followed by nuclease digestion. The first procedure provided evidence that sequences from the large 2300-nucleotide intron (74.6-68.8) were excised and 5' transcripts were spliced to the mRNA body. Utilizing both S1 and Exo VII nucleases, the second procedure demonstrated excision of sequences from the smaller 720-nucleotide intron (68.5-66.3), the splicing of sequences from the second leader (68.8) to the mRNA body, and the formation of an mRNA body of 1700 nucleotides, the size found in vivo. These findings provide evidence that an in vitro system that splices viral RNAs to yield products comparable to those found in vivo is now available.
Coordinate regulation of two cytoplasmic RNA species transcribed from early region 2 of the adenovirus 2 genome.
Early region 2 (E2) of the adenovirus 2 genome specifies a 72,000-dalton DNA-binding protein that is required for viral DNA replication. Electron microscopy studies have detected two major forms of 20S E2 mRNA, one species with a 5' leader from map position 75 and a second form having a leader from position 72 (Chow et al., J. Mol. Biol. 134:265-303, 1979). Only the species with a leader from position 75 was detected at early times; however, both forms were found at late times. We have analyzed the temporal regulation of E2 expression by documenting mRNA accumulation in the cytoplasm. Kinetic studies of pulse-labeled RNAs demonstrated a peak of E2 cytoplasmic RNa synthesis at 10 to 12 h, coinciding with the time of maximal synthesis of the 72,000-dalton DNA binding protein and viral DNA. To estimate the relative abundances of the two major E2 RNA species at various times during infection, total E2 cytoplasmic and polysomal 20S RNAs were isolated by hybridization-selection with specific DNA probes. The leader sequences in the selected RNAs were then quantitated by further RNA-DNA hybridization. We found that the elevated accumulation rate for E2 cytoplasmic RNA at late times reflected an increase in formation of both major species. Moreover, for all time points examined 66% of the mRNA species had a 5' end from map position 75, and 33% had a 5' terminus from position 72. Continuous labeling experiments provided evidence that both RNA forms have comparable half-lives. The results suggest that the two major species encoded by E2 are regulated in a coordinate fashion late in infection.
In vitro processing of intervening sequences in the precursors of messenger ribonucleic acid for adenovirus 2 deoxyribonucleic acid binding protein.
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Splicing patterns of nuclear precursors to the mRNA for adenovirus 2 DNA binding protein.
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Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution.
A method is described for the preparation isolation of highly purified adenovirus RNA species. Cytoplasmic RNAs from cells infected with adenovirus 2 were selected by hybridization to viral DNA fragments bound to nitrocellulose membranes. A series of washes at elevated temperatures (50-70 degrees) determined conditions at which the true hybrids were stable but non-specific RNA was removed. This temperature has been found to correlate with the base composition of the DNA fragment. After washing at this predetermined temperature, the specific RNA was eluted at 85 degrees. The purity of the eluted RNA was greater than 95% as determined by size, sequence specificity, and template activity in an in vitro protein synthesizing system. The method described should be generally useful for purification of specific RNAs.
Size distribution of polyadenylated adenovirus 2 RNA synthesized in isolated nuclei.
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Methylation of ribosomal proteins in HeLa cells.
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