PubMed Health⌕ Search

Biomedical subjects

P Goswami

Publications and source records attributed to P Goswami.

At least 19 recordsLinked to original sources
PubMed1988

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).

Adenosine Triphosphate↗
PubMed1987

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.

Animals↗