The crystal structure of the RNA-binding domain of the small nuclear ribonucleoprotein U1A bound to a 21-nucleotide RNA hairpin has been determined at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the beta-sheet as an open structure, and the AUUGCAC sequence of the loop interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. These interactions include stacking of RNA bases with aromatic side chains of proteins and many direct and water-mediated hydrogen bonds. The structure reveals the stereochemical basis for sequence-specific RNA recognition by the RNP domain.

The crystal structure of the RNA binding domain of the U1 small nuclear ribonucleoprotein A, which forms part of the ribonucleoprotein complex involved in the excision of introns, has been solved. It contains a four-stranded beta sheet and two alpha helices. The highly conserved segments designated RNP1 and RNP2 lie side by side on the middle two beta strands. U1 RNA binding studies of mutant proteins suggest that the RNA binds to the four-stranded beta sheet and to the flexible loops on one end.

Pre-mRNA splicing proceeds by two consecutive trans-esterification reactions via a lariat-intron intermediate. We present the 3.8Å cryoEM structure of the spliceosome immediately after lariat formation. The 5’-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 snRNA triplex, and the 5’-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2’OH. The 5’-exon is held between the Prp8 N-terminal and Linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5’-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step one factors Yju2 and Cwc25 stabilise docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 RT and Linker domains and extends towards Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.

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U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryoEM structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 snRNAs. The structure reveals striking interweaving interactions of the protein and RNA components including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5′-splice site during catalytic activation, forms a hairpin stabilised by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in Prp8’s N-terminal domain. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.