Related Experiment Videos
Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions
Abstract:
Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Murine sarcoma virus ts110 (MuSVts110) exhibits temperature-dependent transformation due to gag-mos gene fusion. A 1.5 kb deletion in MuSVts110 creates an out-of-frame gag-mos junction, while splicing in the 3.5-kb RNA restores the reading frame for transformation.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Murine sarcoma virus ts110 (MuSVts110) exhibits temperature-dependent cellular transformation.
- At permissive temperatures (33°C), MuSVts110-infected cells transform and produce P85gag-mos, while at non-permissive temperatures (39°C), cells appear normal and produce P58gag.
- Previous studies suggested a deletion and potential splicing event involving the gag and mos genes contribute to these phenotypes.
Purpose of the Study:
- To elucidate the precise molecular mechanisms underlying the gag-mos gene fusion and its role in MuSVts110-mediated transformation.
- To characterize the nucleotide sequences and splicing events involved in the formation of MuSVts110 viral RNAs and proteins.
Main Methods:
- Primer extension sequencing of MuSVts110 4-kb and 3.5-kb RNAs to analyze gag-mos gene junctions.
- Comparison of MuSVts110 sequences with wild-type MuSV-349 sequences.
Main Results:
- A 1,488-base deletion in MuSVts110 4-kb RNA fuses gag to mos out-of-frame, resulting in premature termination codons and production of P58gag.
- Analysis of the 3.5-kb RNA revealed a 431-base excision bounded by splice donor and acceptor sites, fusing gag to mos in-frame.
- This splicing event in the 3.5-kb RNA bypasses termination codons and allows translation of the full-length P85gag-mos fusion protein.
Conclusions:
- The temperature-dependent transformation of MuSVts110 is directly linked to alternative splicing of the gag-mos transcript.
- Splicing of the 3.5-kb RNA restores the reading frame, enabling the synthesis of the transforming P85gag-mos protein.
- The study clarifies the genetic basis of MuSVts110 oncogenesis at the molecular level.