The efficacy of two different types of crosslinking approaches for mapping specific RNA-RNA and RNA-protein contacts in complex macromolecular assemblies has recently been demonstrated. These will be applied to gain insight into the mechanism of action of two of the HIV-encoded proteins whose structures are under analysis by other members of this program: the reverse transcriptase (RT) and the Rev protein. The structure of the HIV RT has recently been solved to 3.2angstroms resolution in T. Steitz's laboratory. Model building suggests how the human tRNA3Lys primer might interact with the p66 and p51 subunits, placing its 3' end in the polymerase active site hybridized to the genomic RNA template. To verify the proposed tRNA-enzyme interactions, a series of tRNA3Lys transcripts will be synthesized which contain a single 4- thiouridine preceded by a single 32P-phosphate group. After incubation of these tRNAs with reverse transcriptase in transcription reactions, irradiation at > 300nm light specifically activates the 4-thioU, generating RNA-protein crosslinks. Complete digestion with P1 nuclease (which creates 5' phosphates) then transfers label to the RT subunits. Once various positions in the tRNA have been identified as contacting the p66 or p51 subunit, the analyses can be extended to map crosslinked peptides/amino acids, and also to map template-RT crosslinks. The HIV Rev protein has been proposed to facilitate the export of unspliced RNAs containing the rre binding site by dissociating partially assembled spliceosomes, based primarily on results from in vitro systems, where very high concentrations of Rev must be utilized to inhibit splicing. The effect of Rev on spliceosome assembly in vivo will be examined using a Drosophila tissue culture system (SL2 cells) which exhibits Rev-dependent export of env RNA expressed from an inducible promoter. In vivo-psoralen crosslinking will identify which snRNAs are associated with hybrid-selected env RNA from cells in the absence and presence of Rev. Use of specific probes to select snRNAs, has allowed detection of splicing-dependent crosslinks between a pre-mRNA substrate and U1,U2,U5,U6 and U2/U6 snRNAs and the development of methods for mapping the crosslinked sites. Similar analyses of the in vivo env RNA-snRNA crosslinks should identify the point at which Rev acts to abort spliceosome assembly.
Showing the most recent 10 out of 42 publications
