The small nuclear RNAs (snRNAs) of the U-series (U1, U2, U3,...U10) comprise an abundant and evolutionarily conserved class of RNA molecules present in the nuclei of higher organisms. There is now substantial evidence indicating that the snRNAs are involved at various stages in the processing of pre-messenger RNAs (and pre- ribosomal RNAs) to their mature forms prior to their export to the cytoplasm. Although the snRNAs (with one exception) are synthesized by RNA polymerase II, the genes that code for the snRNAs lack TATA and CCAAT boxes in their 5'-flanking DNA. Therefore, unique DNA sequences and protein factors may be involved in their expression. In previous work in this laboratory, genes coding for chicken U1, U2, and U4 RNAs have been cloned and sequenced. Accumulated evidence indicates that two separate upstream regions of conserved DNA sequence are required for proper promoter function: (1) a proximal region approximately 55 base pairs upstream from the transcriptional initiation site, and (2) a distal region located approximately 200 base pairs upstream of the start site. In this proposal, the DNA sequence requirements of U1 and U4 RNA gene expression will be examined at a fine level by introducing small deletion, insertion, and point mutations into the regulatory regions of the 5'-flanking DNA. The relative expression efficiencies from the wild type and mutant promoters will be measured by transient expression in microinjected frog oocytes and in transfected tissue culture cells. A cell free system that accurately transcribes snRNA genes will be developed. Sequence-specific DNA binding proteins that recognize the regulatory sequences will be purified from chicken tissues, and experiments will be done to demonstrate that these protein factors can stimulate the accurate and efficient expression of snRNA genes in a cell free transcription system. Next, the genes for these transcription factors will be cloned, and then used to synthesize wild type and altered proteins to study the mechanisms by which the proteins activate transcription. Finally, the effect of the transcription factors on chromatin structure will be investigated. One of the clone U4 RNA genes codes for a previously unknown sequence variant of U4 RNA. The potential role of this variant U4 RNA in alternative splicing of mRNA precursors will be examined.
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