Biomedical subjects

B M Bhat

Publications and source records attributed to B M Bhat.

25 records · Page 2Linked to original sources

PubMed1986

Deletion mutants that alter differential RNA processing in the E3 complex transcription unit of adenovirus.

Region E3 of the adenovirus encodes about ten overlapping mRNAs (a to j) with different splicing patterns and with two RNA 3' end sites termed E3A and E3B. We have examined how deletions in 12 viable virus mutants affect differential RNA processing in E3. We assayed E3 mRNAs by the nuclease-gel and RNA blot procedures. Some deletions had no effect whereas others (e.g. deletion of a 3' splice or the E3A 3' end signal) had the anticipated effects on RNA processing. However, deletions in two regions had surprising effects. Deletions in one region (nucleotides 1691 to 2044) enhanced splicing at the upstream 951 5' splice site and the downstream 2157 and/or 2880 3' splice sites. Some of these deletions prevented RNA 3' end formation at the downstream E3A site. Deletion in the other region (nucleotides 2173 to 2237) enhanced an upstream splice site (951 to 2157) such that almost all pre-mRNA was processed into mRNA f. We suggest that these two regions contain cis-acting signals that regulate differential RNA processing. We discuss the results in terms of RNA folding and scanning models for splicing, as well as models for differential RNA 3' end formation at the E3A versus the E3B site.

Adenoviridae↗

PubMed1986

Evidence that AGUAUAUGA and CCAAGAUGA initiate translation in the same mRNA region E3 of adenovirus.

We described a simple method to introduce site-specific mutations into region E3 of adenovirus (Ad). Mutations are made in cloned Ad2 EcoRI-D (map position 76-83), then ligated between Ad5 EcoRI-A (map position 0-76) and EcoRI-B (map position 83-100) to complete the viral genome. We have used this method to isolate a viable virus mutant (dl702) that is relevant to the problems of translation initiation and gene organization in the E3 complex transcription unit. mRNA a in region E3 encodes an abundant glycoprotein termed gp19K. There are two AUGs in mRNA a that are 5' to AUG1204 which initiates gp19K. One of these, AUG1022, could initiate a 6.7K protein, although this protein has not been identified in infected cells. Mutant dl702 has a deletion such that the 6.7K gene is fused in-frame to the gp19K gene. We report that the 6.7K-gp19K fusion protein is synthesized both in dl702-infected cells and after cell free translation of infected cell RNA. The quantity of fusion protein made is much less than that of wild type gp19K. The sequence context of AUG1022 for 6.7K is AGUAUAUGA, and that of AUG1204 for gp19K is CCAAGAUGA. The consensus sequence of eukaryotic initiation codons is CCPuCCAUGG, with the Pu at -3 being important (M. Kozak, Nucleic Acids Res. 12, 857-872, 1984). Our results suggest that (i) AUG1022 can initiate translation in vivo and therefore the 6.7K protein probably is made in infected cells, (ii) that mRNA a is a dicistronic mRNA encoding the 6.7K and gp19K proteins, and (iii) that the initiation codon for 6.7K may be much less efficient than that for gp19K. Thus, the E3 genes may be organized such that the relative abundance of the 6.7K and gp19K proteins is controlled by the efficiency of their initiation codons in the same mRNA.

Adenoviruses, Human↗

PubMed1986

Adenovirus mutants with splice-enhancing mutations in the E3 complex transcription unit are also defective in E3A RNA 3'-end formation.

Region E3 of adenovirus encodes about 10 overlapping mRNAs with different spliced structures. The mRNAs are 5' coterminal and form two major 3'-coterminal families termed E3A and E3B. As a group, the mRNAs have two 5' splice sites and four or five 3' splice sites. We previously described a novel class of virus mutants with deletions that enhance distant upstream and downstream 5' and 3' splice sites in region E3 (S. L. Deutscher, B. M. Bhat, M. H. Pursley, C. Cladaras, and W. S. M. Wold, Nucleic Acids Res. 13:5771-5788, 1985). We now report that two of these mutants, dl710 and dl712, are defective in RNA 3'-end formation at the E3A site. This result was surprising because the deletions in dl710 and dl712 are upstream of the putative signal for E3A RNA 3'-end formation. The explanation that we favor for this result is that the enhanced splicing activity in these mutants results in the splicing out of the E3A 3'-end site from the RNA precursor before the E3A 3' ends have a chance to form.

Adenoviridae↗

PubMed1986

Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive infection by RNA-processing mutants.

Region E3 of adenovirus encodes about nine overlapping mRNAs (a to i) with different spliced structures and with two major RNA 3' end sites termed E3A and E3B. Synthesis of E3 mRNAs was examined by the nuclease-gel procedure at early and late stages of infection by wild-type virus (rec700) and by several E3 deletion mutants. Our results, together with those obtained by electron microscopy (L. T. Chow, T. R. Broker, and J. B. Lewis, J. Mol. Biol. 134:265-303, 1979), suggest that E3 may be differentially regulated at early and late stages at both the promoter and RNA-processing levels. Early after infection, the E3 promoter is used to make mainly mRNAs a and h. Late after infection, the E3 promoter appears to be shut off and the major late promoter is used to make mainly mRNAs d and e. The late L4 mRNA 3' end site is not used early even though early E3 pre-mRNAs transcribe through the L4 RNA 3' end site. The nucleotide 768-951 exon, which is the y leader on many L5 mRNAs, is very abundant late. (Nucleotide +1 is the major E3 transcription initiation site.) Early after infection, the 951 5' splice site is enhanced 5- to 10-fold in the dl712 (delta 1691 to 2122) group of mutants; late after infection, these mutants resemble the wild type. We speculate that the activity of the 951 5' splice site is regulated at early and late stages; it is suppressed early to permit synthesis of mRNA a, and it is activated late to permit synthesis of mRNAs d, e, and L5. With dl719 (delta 2173 to 2237), the 951----2157 splice is enhanced both early and late; we suggest that this deletion enhances the 2157 3' splice site.

Adenoviridae↗

PubMed1985

Novel deletion mutants that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2.

Region E3 of adenovirus is a "complex" transcription unit: i.e. different mRNAs and proteins arise by differential RNA 3' end selection and differential splicing of the primary transcript. We are using viable virus mutants to understand the controls that dictate the specificity and efficiency of the RNA processing signals. We describe a novel class of deletion mutations that enhance a natural 5' splice site located approximately 740 nucleotides (nt) upstream. In particular, deletions within nt 1691-2044 in the E3 transcription unit result in a 5-fold enhancement of the 5' splice site at nt 951 (as reflected in steady-state mRNA). The effect is specific, because the deletions do not affect the 5' splice site at nt 372, and because deletions within nt 2044-2214 do not affect either the 951 or the 372 5' splice sites. As a consequence of the enhanced splicing at the 951 5' site, synthesis of the major E3 mRNA and the major E3 protein (gp19K) are dramatically reduced. At least one of the natural 3' splice sites, located at nt 2157, is the recipient of the enhanced splicing at the 951 5' splice site. We conclude that sequences located within nt 1691-2044 affect (probably in cis) splicing at the 951 5' splice site. We speculate that nt 1691-2044 includes a splicing control region which functions to suppress splicing at the 951 5' splice site.

Adenoviruses, Human↗

PubMed1985

ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus.

We mapped the location of the E3A RNA 3' end site in the E3 transcription unit of adenovirus 2. The procedure used was nuclease-gel analysis with 32P-labeled RNA probes. The poly(A) addition sites were microheterogeneous and were located approximately 17 to 29 nucleotides downstream from an ATTAAA sequence. To identify the sequences that make up the E3A RNA 3' end signal, we constructed five viable virus mutants with deletions in or near the E3A RNA 3' end site. The mutants were analyzed for E3A RNA 3' end formation in vivo. No effect was observed from a 47-base-pair (bp) deletion (dl716) or a 72-bp deletion (dl714) located 22 and 19 nucleotides, respectively, upstream of the ATTAAA. In contrast, E3A RNA 3' end formation was abolished by a 554-bp deletion (dl708) that removes both the ATTAAA and the poly(A) addition sites, a 124-bp deletion (dl713) that removes the ATTAAA but leaves the poly(A) addition sites, and a 65-bp deletion (dl719) that leaves the ATTAAA but removes the poly(A) addition sites. These results indicate that the ATTAAA, as well as downstream sequences, including the poly(A) addition sites, are required for E3A RNA 3' end formation.

Adenoviruses, Human↗

PubMed1985

Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2.

Five viable virus mutants were constructed with deletions near a 3' splice site located at nucleotide 2157 in the E3 transcription unit of adenovirus 2. The mutants were examined for splicing activity at the 2157 3' splice site in vivo by nuclease-gel analysis of steady-state cytoplasmic mRNA. Splicing was not prevented by an exon deletion (dl719) that leaves 16 5'-proximal exon nucleotides intact or by intron deletions that leave 34 (dl717, dl712) or 18 (dl716) 3'-proximal intron nucleotides intact. The sequences deleted in one of these intron mutants (dl716) include the putative branchpoint site used in lariat formation during splicing. Thus, a surrogate branchpoint site apparently can be used for splicing. Another intron mutant (dl714) has a deletion that leaves 15 3'-proximal intron nucleotides intact; remarkably, this deletion virtually abolished splicing, even though the deletion is only 3 nucleotides closer to the splice site than is the deletion in dl716 which splices normally. The three nucleotides deleted in dl714 that are retained by dl716 are the sequence TGT. The TGT sequence is located on the 5' boundary of the pyrimidine-rich region upstream of the nucleotide 2157 3' splice site. Such pyrimidine-rich regions are ubiquitous at 3' splice sites. Most likely, the TGT is required for splicing at the nucleotide 2157 3' splice site. The TGT may be important because of its specific sequence or because it forms the 5' boundary of the pyrimidine-rich region.

Adenoviruses, Human↗