Nucleic Acids Research Nucleotide sequence determination and secondary structure of Xenopus U3 snRNA

Using a combination of RNA sequencing and construction of cDNA clones followed by DNA sequencing, we have determined the primary nucleotide sequence of U3 snRNA in Xenopus laevis and Xenopus borealis. This molecule has a length of 219 nucleotides. Alignment of the Xenopus sequences with U3 snRNA sequences from other organisms reveals three evolutionarily conserved blocks. We have probed the secondary structure of U3 snRNA in intact Xenopus laevis nuclei using single-strand specific chemical reagents; primer extension was used to map the positions of chemical modification. The three blocks of conserved sequences fall within singlestranded regions, and are therefore accessible for interaction with other molecules. Models of U3 snRNA function are discussed in light of these data. INTRODUCTION All eukaryotes examined so far have a distinctive set of uridine-rich small nuclear RNAs [U snRNAs; (1-2)] that are coupled with proteins in ribonucleoprotein particles [snRNPs]. Most U snRNAs are products of RNA polymerase II transcription (3-5). They are distinct from typical RNA polymerase II transcripts in that most snRNAs (except U6) have a unique trimethylguanosine cap at their 5' end (2), and they lack a poly (A) tail at their 3' end. What roles are played by snRNPs? snRNPs containing U1, U2, U4, U5 and U6 snRNAs are required for splicing messenger RNA precursors [reviewed in 6-9]. Early in splicing, the U1 snRNP binds to the 5' splice site of the intron. RNase H cleavage has shown that the 5' end of U1 snRNA is necessary for splicing at the 5' end of the intron (10-12), and a compensatory mutation near the 5' end of U1 snRNA is able to suppress a mutation at the 5' splice site (13), showing that base-pairing between U1 snRNA and the 5' splice site is an important feature of the recognition. Although this implies that the base-pairing of Ul snRNA to the 5' splice site is necessary, it is not sufficient in itself, as Ul snRNP proteins are needed © IRL Press Limited, Oxford, England. 2 1 2 7 Nucleic Acids Research to impart specificity to U1 snRNP binding (14). RNase H cleavage has shown that the snRNA molecules of U2, U4 and U6 are also required for splicing (11-12, 15-16). It has been proposed that the yeast analogue to U2 snRNA (17) hydrogen-bonds to the conserved branch site within yeast introns; this has been supported by compensatory mutations in the yeast U2 snRNA analogue that can suppress mutations in the intron branch site (18). snRNPs may also play other roles in RNA processing in addition to splicing. For example, the U7 snRNP is involved in processing the 3' end of histone mRNA, and compensatory mutation experiments support the proposal that U7 snRNA base-pairs with a 3' end processing signal in the histone mRNA precursor (19). A function has not been identified for the U3 snRNP. Does U3 snRNA also play a role in RNA processing? The U3 snRNP is different from the other snRNPs because it is localized in the nucleolus ( 1 , 2), where it is associated with pre-ribosomal particles greater than 60S (20). Because of these features, it seemed possible that U3 snRNA might be important for pre-rRNA processing (a nucleolar event), perhaps in removal of the internal transcribed spacer 2 (ITS 2) during the processing of 32S pre-rRNA to 28S rRNA (21-23). This processing event releases 5.8S RNA, the counterpart of the 5' end of prokaryotic 23S rRNA (24-26). The notion that the 5.8S gene became separated from the main body of the 28S gene during evolution by the insertion of the ITS 2 prompted an analogy between ITS 2 and introns, including the speculation that a region within U3 snRNA base-pairs with a sequence at the 5' end of the ITS 2 of vertebrates (22, 23). At the time this model was proposed, the only species in which both the ITS 2 and U3 snRNA sequences were known was the rat. Although the list has now grown slightly (Table I ) , there is a taxonomic gap between mammals and lower eukaryotes. Moreover, the 5 end of the ITS 2 is not conserved in the latter group (23). To test the hypothesized interaction of U3 snRNA with ITS 2, we have extended the available data base by examining a non-mammalian vertebrate. We chose the amphibia Xenopus laevis and Xenopus borealis because previously Furlong and Maden (32) had compared the ITS 2 sequences from these two species. They identified conserved tracts interspersed among divergent sequences. We predicted that if any of the conserved tracts in ITS 2 base-pair with U3 snRNA, then both X. laevis and X. borealis should have an identical sequence in U3 snRNA that is complementary to the conserved tract of ITS 2. Alternatively, if mutation has occurred, then compensatory base changes should be found between a non-conserved stretch