S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus

Abstract

The structures of murine sarcoma virus (MuSV) tsllO viral RNA and intracellular RNA present in MuSV tsllO-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV tsllO viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV tsllO RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed Al) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and plO coding sequences. The 3' border of the Al deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5kb MuSV tsllO RNA, the 5' border of the deleted central region (termed A2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV tsllO RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV tsllO RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33°C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39°C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33°C permits the reappearance of the 3.5-kb RNA at its original level.

Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSVts110

“…1). We have previously shown that the spliced 3.5-kb RNA will protect only 120 bases of the DD.29 DNA (11) (Fig. 4, lane 4), establishing that the gag gene in this RNA extends only up to the 5' splice site at nucleotide 2,017.…”

“…4, lane 4), establishing that the gag gene in this RNA extends only up to the 5' splice site at nucleotide 2,017. In contrast, the unprocessed 6m2 4.0-kb RNA will fully protect the DD.29 DNA fragment (11) (Fig. 4, lane 6).…”

“…originally described and used by Nash et al 1984 (12) and will be briefly described here for clarity ( Fig. 1).…”

“…S1 nuclease analysis. These analyses were done essentially as described previously (11,12). Briefly, 5' end-labeled AvaI-digested pBA.36 DNA was hybridized for 3 h to 10 pug of total cellular RNA from 6m2 cells grown at 39, 33, or 28°C…”

“…Splicing excises a 431-base intron that contains an out-of-frame gag-mos junction, replacing it with a shortened but continuous gag-mos open reading frame (12). For reasons that are still unclear, splicing of the MuSVtsllO 4.0-kb viral RNA is thermosensitive; this processing event only occurs at growth tempera-tures between 28 and 33°C (5,11). Because the splicing event effectively regulates the production of the P85ag-mostransforming protein, 6m2 cells appeared transformed if grown at 28 to 33°C and normal if grown at 39°C.…”