We are continuing to characterize the biosynthesis and function of the human small RNA species U1, U2, and 7SL. U1 and U2 small nuclear RNA (snRNA) are components of small nuclear ribonucleoprotein particles (snRNPs) which are involved in RNA processing. The cytoplasmic 7SL RNA is a component of signal recognition particle (SRP) which guides nascent secretory polypeptides to the rough endoplasmic reticulum. Our studies on the transcription and maturation of U1, and U2, and 7SL RNA will lay the groundwork for a mutational analysis of the function of these RNAs in vivo, because we will know which regions in each RNA can be mutagenized without grossly impairing normal biosynthesis of the RNA species. The internal 7SL promoter for RNA polymerase III is interesting because we have found its efficiency to be strongly modulated by 5' flanking DNA both in vitro and in vivo. The U1 and U2 promoters are worth studying because both these unusual RNA polymerase II transcription units lack any obvious upstream TATA box. Formation of the 3' end of U1 and U2 is important because snRNAs, together with histone mRNAs, are the only stable RNA polymerase II products lacking a 3' terminal poly(A) tract. Other laboratories have shown that the 3' end of histone mRNA is formed by RNA processing mediated by a rare snRNP containing a newly described snRNA species. We have found that the 3' ends of both U1 and U2 are also formed by RNA processing, although the RNA sequences are very different from those of histone mRNA. We are currently using four different assay systems to examine U1, U2, and 7SL biosynthesis: (1) microinjection into Xenpous oocytes of human genes mutagenized in vitro provides a rapid and convenient assay, but may run afoul of species-specific differences in transcription and processing between Xenpous and man; (2) transient expression of mutagenized human genes introduced into HeLa cells with an SV40-based vector represents a slightly less convenient assay, but assures us of studying the genes in a homologus environment; (3) in vitro, transcription and processing in the HeLa extract developed by Hernandez and Keller (1983) should allow us to characterize not just the DNA and RNA sequences which are critical for biosynthesis, but also the protein factors which interact with these sequences; and finally (4) we are further analyzing U1 and U2 maturation by microinjection of Xenopus oocytes with labelled runoff transcripts of mutagenized U1 and U2 genes transcribed in vitro under control of a bacteriophage promoter.
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