The spliceosome is a dynamic assembly of five small nuclear ribonucleoproteins (snRNPs) that removes introns from eukaryotic pre-mRNA. U6 is the most conserved of the spliceosomal snRNAs and participates directly in catalysis. Here, we report the crystal structure of the Saccharomyces cerevisiae U6 snRNP core, containing most of U6 snRNA and all four RRM domains of the Prp24 protein. It reveals a unique interlocked RNP architecture that sequesters the 5′ splice site-binding bases of U6 snRNA. RRMs 1, 2 and 4 of Prp24 form an electropositive groove that binds double-stranded RNA and may nucleate annealing of U4 and U6 snRNAs. Substitutions in Prp24 that suppress a mutation in U6 localize to direct RNA-protein contacts. Our results provide the most complete view to date of a multi-RRM protein bound to RNA, and reveal striking co-evolution of protein and RNA structure.

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

Pre‐mRNA splicing is a key step in eukaryotic gene expression that is catalyzed by the spliceosome. The spliceosome contains 5 RNA‐protein complexes called small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs). Assembly of the spliceosome from these snRNPs involves numerous complex and dynamic RNA‐protein interactions. The U6 snRNP is comprised of the U6 small nuclear RNA (snRNA) bound to proteins Prp24 and the Lsm2‐8 heteroheptameric ring, the latter of which binds to the 3′ end of U6. Previous research in our laboratory determined the crystal structure of a large fragment of U6 snRNA bound to the four RNA recognition motifs (RRMs) in Prp24. As an extension of that work, we are now investigating the interaction of the Lsm2‐8 ring complex with the 3′ ‐end of U6, using native polyacrylamide gel electrophoresis (PAGE). Previously published data indicated that the 3′ end of yeast U6 snRNA contains either a free 3′ hydroxyl or 3′ phosphate group, whereas most other organisms have a 2′,3′ cyclic phosphate at the end of U6 snRNA. Our preliminary native PAGE data demonstrate that yeast U6 with a free 3′ hydroxyl end has a significantly higher binding affinity for the yeast Lsm2‐8 complex than the same RNA with a cyclic phosphate. We hypothesize that a free 3′ hydroxyl group may be important for high affinity binding of the Lsm2‐8 complex and subsequent crystallization of the yeast U6 snRNP. Attempts to crystallize the complete U6 snRNP are currently underway.