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J. Virol., Dec 1995, 7724-7733, Vol 69, No. 12
Copyright © 1995, American Society for Microbiology

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

JW Touchman, I D'Souza, CA Heckman, R Zhou, NW Biggart and EC Murphy Jr
Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.

Balanced splicing of retroviral RNAs is mediated by weak signals at the 3' splice site (ss) acting in concert with other cis elements. Moloney murine sarcoma virus MuSVts110 shows a similar balance between unspliced and spliced RNAs, differing only in that the splicing of its RNA is, in addition, growth temperature sensitive. We have generated N- nitroso-N-methylurea (NMU)-treated MuSVts110 revertants in which splicing 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 thermosensitivity. In a representative revertant (NMU-20), we found that complete splicing was conferred by a G-to-A substitution generating a consensus branchpoint (BP) signal (-CCCUGGC- to -CCCUGAC- [termed G(-25)A]) at -25 relative to the 3' ss. Weakening this BP to -CCCGAC- [G(-25)A,U(-27)C] moderately reduced splicing at the permissive temperature and sharply inhibited splicing at the originally nonpermissive temperature, arguing that MuSVts110 splicing thermosensitivity depends on a suboptimal BP-U2 small nuclear RNA interaction. This conclusion was supported by results indicating that lengthening the short MuSVts110 polypyrimidine tract and altering its uridine content doubled splicing efficiency at permissive temperatures and nearly abrogated splicing thermosensitivity. In vitro splicing experiments showed that MuSVts110 G(-25)A RNA intermediates were far more efficiently ligated than RNAs carrying the wild-type BP, the G(- 25)A,U (-27)C BP, or the extended polypyrimidine tract. The efficiency 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 retroviruses and, in addition, that its splicing thermosensitivity is a response to the presence of multiple suboptimal splicing signals.

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