U6 small nuclear RNA (U6 snRNA) is a component of eukaryotic spliceosomes and is required for splicing of nuclear pre-messenger RNAs. It has been suggested that U6 RNA may be directly involved in catalysis during nuclear pre-mRNA splicing. U6 RNA, besides being the most conserved of all U snRNAs, is unique in many respects. First, it contains CH3-O-pppG cap instead of the trimethylguanosine (TMG) cap found in other U snRNAs. Second, while TMG cap-containing U snRNAs are transcribed by RNA polymerase II, U6 RNA is transcribed by pol III. Third, the transcription of mammalian U6 snRNA gene does not require any internal promoter and the transcription is dependent on 5' flanking sequences. Therefore, studies on U6 snRNA synthesis are important to understand the fundamental cellular events. Experiments will be carried out with the following specific objectives: (A). To purify the factors involved in the capping of U6 and 7SK small nuclear RNAs. (B). To study the mechanism of capping with purified components. Since U6 RNA has an unusual cap structure (gamma-monomethyl phosphate), it offers a model system towards elucidating the fundamental differences in the mechanism(s) of cap formation on U6 and 7SK snRNAs vs mnRNAs and other U snRNAs. (C). To obtain monoclonal and polyclonal antibodies specific to mepppG and mepppA cap structures. (D). To identify and characterize the minor methyl-capped snRNAs. (E). To identify and purify the factor(s) involved in the transcription of U6 snRNA gene.
