Abstract
U6 RNA is an abundant, capped small nuclear RNA (snRNA) associated with hnRNP particles (Reddy, R., and Busch, H. (1983) Prog. Nucleic Acid Res. Mol. Biol. 30, 127-162). Small nuclear ribonucleoprotein particles containing U4 and U6 RNAs are required components for splicing of pre-mRNAs (Berget and Robberson, 1986; Black and Steitz, 1986). In this study the Drosophila U6 RNA genes have been isolated and characterized. The Drosophila genome contains three U6 snRNA genes which are clustered in a 2-kilobase-pairs long DNA fragment. The U6 RNA coding regions are 100% homologous in all three genes, but the flanking sequences diverged significantly from each other. A possible secondary structure model for the Drosophila U4/U6 RNA complex is presented. Consistent with our previous observation that U6 RNA is a RNA polymerase III product (Reddy, R., Henning, D., Das, G., Harless, M., and Wright, D. (1987) J. Biol. Chem. 262, 75-81), all three genes contained a region homologous to the consensus intragenic regulatory region and a cluster of T residues on the 3'-end, characteristic of genes transcribed by RNA polymerase III. A TATA box was found between nucleotides -23 and -31, and a stretch of 28 nucleotides from -43 to -71 was conserved in the 5'-flanking region of all three U6 RNA genes. The Drosophila U6 RNA genes were transcribed in vitro by Drosophila nuclear extracts but were not transcribed by Novikoff hepatoma or HeLa cell extracts. Similarly, a mouse U6 RNA gene was transcribed in Novikoff hepatoma or HeLa cell extracts but not in Drosophila nuclear extracts. These results suggest that species-specific factor(s) are involved in the transcription of U6 snRNA genes.
Highlights
Inthis Recently, it was shown that U4/U6 RNP complex is required study theDrosophila U6 RNA genes have been isolatedfor the splicing of pre-mRNAs
The Drosophila Genome Contains Three U6 RNA Genes-Isolation of U6Gene Containing Recombinant Phuges- Southern hybridization of Drosophila genomic DNA, using Drosophila genomic library was screened with 3'-end-labeled conditions and probe identicalto thoseemployed in analyzing rat U6 RNA
Drosophila U4 snRNA is >75%homologous torat U4 snRNA and Drosophila U4 snRNA geneswere shown to hybrid select rat U4 snRNA (Saba et al, 1985).These results show that U4 RNA gene(s) are not immediately .adjacent to the U6 RNA genes in the Drosophila genome
Summary
Isolation of U6Gene Containing Recombinant Phuges- Southern hybridization of Drosophila genomic DNA, using Drosophila genomic library was screened with 3'-end-labeled conditions and probe identicalto thoseemployed in analyzing rat U6 RNA. DU6-1 contained TATATATAG sequence a t -31 to -23; DU6-2 contained TTTATATAG at -30 to -22; DU6-3 contained TATATATAG sequence at -30 to -22; and mouse U6 RNA gene contained TATAAATAT sequence at -31 to -22 These results show that the DNA sequence corresponding to U6 RNA is conserved in Drosophila U6 RNA genes butthe flanking regions appear to be divergent, except for some conserved upstream sequences (Fig. 3). The U6 RNA synthesized in vitro initiated properly with pppG at nucleotide 1,andthis pppG was converted in vitro into XpppG cap structure (oligonucleotide, Fig. 5) These data showed that U6 RNA transcribed from Drosophila U6 genes wasproperly initiated resulting in an RNA of the right size.
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