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We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.Although the U5 small nuclear ribonucleoprotein (snRNP) is an essential cofactor in pre-mRNA splicing, its precise function is unknown. The U5 snRNP has long been hypothesized to play a role in the second catalytic step of splicing, perhaps as a factor required for the recognition of the 3' splice site (6,28,38). Indeed, we have recently identified several proteins that genetically interact with the U5 small nuclear RNA (snRNA) and are required for the second step of splicing (8). One of these proteins, the product of the SLU7 gene, functions in the selection of distant 3' splice sites (7). Whether any of these proteins is an integral U5 snRNP protein remains an open question; nevertheless, they provide strong evidence linking U5 function to the recognition and utilization of the 3' splice site. Moreover, several studies indicate that conserved sequences in the U5 snRNA, itself, can interact with exon sequences at the intron-exon junctions. Newman and Norman (25) have demonstrated that mutations in the Saccharomyces cerevisiae U5 snRNA can suppress mutations in both 5' and 3' splice sites, suggesting that the U5 snRNA can form hydrogen bonds with both 5' and 3' exon sequences. In agreement with this finding, a subset of the U5 nucleotides implicated in the 5' splice site-U5 interaction have been cross-linked to exonic and intronic RNA sequences near the 5' splice site, indicating a close, physical juxtaposition of U5 and 5' splice site nucleotides during splicing (41, 44). Taken together, these studies suggest that the U5 snRNA plays an active role in the function of this snRNP.Aside from the few nucleotides implicated in the exon-U5 interaction, only the conserved Sm ...

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We describe cloned segments of rDNA that contain short type I insertions of differing lengths. These insertions represent a coterminal subset of sequences from the right hand side of the major 5kb type I insertion. Three of these shorter insertions are flanked on both sides by a short sequence present as a single copy in uninterrupted rDNA units. The duplicated segment is 7, 14 and 15 nucleotides in the different clones. In this respect, the insertions differ from the 5kb type I insertion, where the corresponding sequence is found only at the right hand junction and where at the left hand side there is a deletion of 9 nucleotides of rDNA (Roiha et al.,1981). One clone is unusual in that it contains two type I insertions, one of which is flanked by a 14 nucleotide repeat. The left hand junction of the second insertion occurs 380 nucleotides downstream in the rDNA unit from the first. It has an identical right hand junction to the other elements and the 380 nucleotide rDNA sequence is repeated on both sides of the insertion. We discuss the variety of sequence rearrangements of the rDNA which flank type I insertions.

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