Crystal Structure of a Core Spliceosomal Protein Interface

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 103; no. 5; pp. 1266 - 1271
• Main Authors: Schellenberg, Matthew J., Edwards, Ross A., Ritchie, Dustin B., Kent, Oliver A., Golas, Monika M., Stark, Holger, Lührmann, Reinhard, Glover, J. N. Mark, MacMillan, Andrew M.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 31-01-2006; National Acad Sciences
• Subjects: Adenosine - chemistry; Alternative Splicing; Amino Acid Sequence; Amino acids; Biological Sciences; Cross-Linking Reagents - pharmacology; Crosslinking; Cryoelectron Microscopy; Crystallography, X-Ray; Data collection; Humans; Introns; Messenger RNA; Microscopy, Electron; Models, Molecular; Molecular Sequence Data; Nucleotides; Peptides - chemistry; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Proteins - chemistry; Ribonucleoprotein, U2 Small Nuclear - chemistry; Ribonucleoproteins, Small Nuclear - chemistry; RNA; RNA - chemistry; RNA, Messenger - metabolism; RNA-Binding Proteins - chemistry; Small nuclear ribonucleoproteins; Spliceosomes; Spliceosomes - chemistry; Spliceosomes - metabolism; Splicing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0508048103

The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-Å crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal α-helix and a portion of the SF3b155 peptide. An analysis of RNA-protein crosslinking to wildtype and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as modelbuilding by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14·SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.