Activation of cryptic splice sites in murine sarcoma virus-124 mutants

Abstract

We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVtsllO retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVtsllO or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site, whereas a mutated branchpoint activated less efficient but thermosensitive splicing.

Branchpoint and polypyrimidine tract mutations mediating the loss and partial recovery of the Moloney murine sarcoma virus MuSVts110 thermosensitive splicing phenotype

“…Consequently, MuSVts110-infected cells remain morphologically normal at physiological temperatures and above (21,23,35,36). Our previous studies have shown that MuSVts110 RNA splicing is cis regulated in part by the strength of the BP, since introduction of a ␤-globin BP into a splicing-negative mutant of MuSVts110 activated splicing that was both very efficient and temperature insensitive (11), and also by a negative regulator in the second exon (the exon 2 distal element [E2DE]) (12,56). Removal of the E2DE relaxes the interference with splicing observed at higher growth temperatures without producing a pronounced enhancement of splicing at the lower temperatures (56).…”

Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression

“…The effect of this deletion on viral gene expression is to prohibit expression of the v-mos gene and activate previously cryptic splice sites in the gag and v-mos genes for growth temperature-restricted splicing (27,28). We have previously established that this deletion is the major, if not sole, determinant of the unusual phenotype exhibited by MuSVtsllO (9,10). This was most clearly shown by assessing gene expression in an MuSV-124 mutant (termed ts32) engineered to contain no other deviations from the wild-type sequence except a 1,487-nucleotide deletion in precisely the same location as found in MuSVtsllO RNA.…”

“…First, none of these sequence differences appear to be near or affect any conserved splicing signals. Second, no sequence changes were observed in the two regions demonstrated to influence the thermosensitivity of MuSVtsllO RNA splicing, the MuSVtsllO branch point (6,10) and the 3' half of the v-mos exon (34). In addition, since we have previously shown that translation of the MuSVts1lO gag gene is not required to maintain the MuSVtsllO splicing phenotype (11), it would follow that neither of the substitutions observed in the gag gene would be expected to affect splicing.…”

“…To sidestep this problem, we constructed from wild-type MuSV-124 DNA a viral DNA (termed ts32 DNA) containing precisely the same 1,487-nucleotide deletion found in authentic MuSVtsllO RNA (9). Several subsequent studies indicated that all of the known characteristics of MuSVtsllO (e.g., the restriction of cell transformation, viral RNA splicing, and production of P85fag-mos to low growth temperatures) were conserved in ts32, arguing that the 1,487-nucleotide deletion was the major, if not sole, determinant of the novel pattern of viral RNA splicing and gene expression exhibited by MuSVtsllO (7,9,10). Moreover, the conditional splicing of ts32 RNA was shown to be regulated in cis by transcript structure (10, 11).…”

Nickel mutagenesis: Alteration of the MuSVts 110 thermosensitive splicing phenotype by a nickel‐induced duplication of the 3′ splice site

“…To locate and characterize the nickel-induced mutation, we amplified portions of the integrated MuSVtsl 10 DNA in N1 cells by PCR [28]. In other work, we have determined that MuSVtsllO RNA splicing is regulated in cis by intron structure [24,29] as well as by exonic regions extending for several hundred bases from each splice site (D. A. Stemer and E. C. Murphy, Jr., unpublished data). Consequently, the MuSVtsllO intron and sections of the flanking exons in both N1 and control 6m2 cellular DNA were amplified by PCR using the SUZ1 and SUZ4 primers ( Figure 5A).…”