BRANCHPOINT AND POLYPYRIMIDINE TRACT MUTATIONS MEDIATING THE LOSS ANDPARTIAL RECOVERY OF THE MOLONEY MURINE SARCOMA-VIRUS MUSVTS110 THERMOSENSITIVE SPLICING PHENOTYPE

Balanced splicing of retroviral RNAs is mediated by weak signals at th e 3' splice site (ss) acting in concert with other cia elements. Molon ey murine sarcoma virus MuSVts110 shows a similar balance between unsp liced and spliced RNAs, differing only in that the splicing of its RNA is, in addition, growth temperature sensitive. We have generated N-ni troso-N-methylurea (NMU)-treated MuSVts110 revertants in which splicin g was virtually complete at all temperatures and have investigated the molecular basis of this reversion on the assumption that the findings would reveal cis-acting elements controlling MuSVts110 splicing therm osensitivity. In a representative revertant (NMU-20), we found that co mplete splicing was conferred by a G-to-A substitution generating a co nsensus branchpoint (BP) signal (-CCCUG (G) under bar GC- to -CCCUG (A ) under bar AC- [termed G(-25)A]) at -25 relative to the 3' ss. Weaken ing this BP to -CCC (C) under bar CGAC- [G(-25)A,U(-27)C] moderately r educed splicing at the permissive temperature and sharply inhibited sp licing at the originally nonpermissive temperature, arguing that MuSVt s110 splicing thermosensitivity depends on a suboptimal BP-U2 small nu clear RNA interaction. This conclusion was Supported by results indica ting that lengthening the short MnSVts110 polypyrimidine tract and alt ering its uridine content doubled splicing efficiency at Permissive te mperatures and nearly abrogated splicing thermosensitivity. In vitro s plicing experiments showed that MuSVts110 G(-25)A RNA intermediates we re far more efficiently ligated than RNAs carrying the wild-type BP, t he G(-25)A,U (-27)C BP, or the extended polypyrimidine tract, The effi ciency of ligation in vitro roughly paralleled splicing efficiency in vivo [G(-25)A BP > extended polypyrimidine tract > G(-25)A,U(-27)C BP > wild-type BP]. These results suggest that MuSVts110 RNA splicing is balanced by cis elements similar to those operating in other retroviru ses and, in addition, that its splicing thermosensitivity is a respons e to the presence of multiple suboptimal splicing signals.