Evans, P

Kambach, C

Li, J

Nagai, K

Oubridge, C

Price, SR

Walke, S

Young, R

UCL Discovery

 	 C40  C40		     	 s8a.m6.o1 Crystallographic dissection of thespliceosomal snRNP structures. K. Nagai, C. Kambach,S. Walke, R. Young, C. Oubridge, S. Price, P. Evans and J.Li. MRC Laboratory of Molecular Biology, Hills Road,Cambridge CB2 2QH, UK.Keywords: proteins, nucleic acids.The protein coding sequence of the majority ofeukaryotic genes is interrupted by non-coding interveningsequences (introns). Following transcription into mRNAprecursors the introns are excised from pre-mRNA to formcontinuous coding sequences by two-step trans-esterifica-tion reactions within a macromolecular assembly called thespliceosome. The major components of the spliceosomeare four large RNA-protein complexes called the U1, U2,U4/U6 and U5 small nuclear ribonucleoprotein particles(snRNPs). These snRNPs contain seven Sm proteins (B/B’,D1, D2, D3, E, F and G) in common, which assemblearound the Sm site present in four of the major spliceoso-mal small nuclear RNAs (snRNAs) and form a globularcore domain.  In addition to the Sm proteins each snRNPcontains some specific protein components. For examplethe U1 snRNP contains three specific proteins, namely U170k, U1A and U1C proteins.  The 12S U2 snRNP containstwo specific proteins, namely the U2B” and U2A’ proteins.We are trying to understand the molecular mechanism ofpre-mRNA splicing through crystallographic, biochemicaland genetic studies of the snRNPs (1).The Sm proteins share a common sequence motif intwo segments, Sm1 and Sm2, separated by a short varibalelinker.  We have determined the crystal structures of twoSm protein complexes, D3B and D1D2 (2). All four Smproteins, D3, B, D1 and D2, have a common fold contain-ing an N-terminal helix followed by a strongly bent fivestranded anti-parallel beta sheet, and the D1D2 and D3Bdimers superpose closely in their core regions, includingthe dimer interfaces.  The crystal structures suggest that theseven Sm proteins could form a closed ring and thesnRNAs may be bound in the positively charged centralhole (2).The U1A protein contains two copies of the RNP motif(or RRM or RBD), one of the most commonly found RNAbinding motifs.  We have determined the crystal structureof the RNA-binding domain of the U1A protein bound to a21-nucleotide RNA hairpin (3). The ten-nucleotide RNAloop binds to the surface of the beta sheet as an openstructure, and the AUUGCAC sequence of the loop inter-acts extensively with the protein.  These interactions in-clude stacking of RNA bases with aromatic side chains ofthe protein and many direct and water-mediated hydrogenbonds. The structure reveals the stereochemical basis forsequence-specific RNA recognition by the RNP domain.We have also determined the crystal structure of a ternarycomplex between the U2B"/U2A' protein complex andhairpin-loop IV of U2 snRNA (4).  Unlike its close homo-logue, the U1A protein, U2B" binds to its cognate RNAonly in the presence of U2A', which contains leucine-richrepeats.  The crystal structure reveals how protein-proteininteraction regulates RNA binding specificity and how re-placing only a few key residues allows the U2B" and U1Aproteins to discriminate between their cognate and non-cognate RNA hairpins by forming alternative networks ofinteractions.Our next goal is to assemble all the protein subunitsand the in vitro transcribed snRNAs to reconstitute the U1and 12S U2 snRNPs and crystallise them for structural de-termination by X-ray crystrallography. This will provideconsiderable insight into the architecture of the snRNPsand will help us understand the molecular mechanism ofpre-mRNA splicing (1).[1] Kambach C, Walke S, Nagai K: Structure and assembly of thespliceosomal small nuclear ribonucleoprotein particles. Curr. Opin. inStructural Biology 1999, 9:222-230[2] Kambach C, Walke S, Young RA, Avis JM, De la Fortelle E, RakerVA, Lührmann R, Li J, Nagai K: Crystal structures of two Sm proteincomplexes and their implications for the assembly of the spliceosomalsnRNPs. Cell 1999, 96:375-387.[3] Oubridge C, Ito N, Evans PR, Teo HC, Nagai K: Crystal strucure ofthe RNA-binding domain of the U1 small nuclear  ribonucleoprotein A.Nature 1994, 348:515-520[4] Price SR, Evans PR, NagaiK: Crystal structureof the spliceosomalU2B"-U2A' protein complex bound to a fragment of U2 small nuclearRNA. Nature 1998, 394:645-650.s8a.m6.o2 Structural studies of  Sm-related proteinsfrom archaea. I. Törõ, C. Mayer, S. Thore, J. Basquin, H.Dreher, M. Dreher, B. Séraphin and D. Suck, EuropeanMolecular Biology Laboratory, Meyerhofstrasse 1,Postfach 102209, 69012 Heidelberg, GermanyKeywords: proteins, nucleic acids.Sm and Sm-like (LSm) proteins are components of thesmall nuclear ribonucleoprotein particles (snRNPs) ofeukaryotic cells which are involved in a variety ofprocesses including pre-mRNA splicing, telomerreplication, rRNA processing and tRNA maturation. Ineukaryotes the core domains of the snRNPs contain sevendistinct members of the Sm protein family associated withan RNA. Recently, Sm-related proteins of unknownfunction were discovered  in several archaeal organismsusing sequence-database searches.  In contrast toeukaryotes, archaea appear to encode only one or two Sm-related proteins. We have undertaken  a structural andfunctional characterization of these proteins.We have cloned and expressed several Sm-relatedproteins from different archaeal sources includingArchaeoglobus fulgidus, Pyrococcus abyssi, andSulfolobus solfataricus. At present three of  these proteinshave been crystallized and the structure of the A.fulgidusprotein AF-Sm2 has been determined by the SIRADtechnique and refined to an R-factor of 20.5% using 1.95Ådata. In addition, the structure of  the P.abyssi protein PA-Sm1 has been solved by molecular replacement.In the crystal AF-Sm2 forms a doughnut-shapedhexamer approx. 60Å in diameter and 30Å high. Theoverall fold of the monomer  - a strongly bent 5-strandedbeta-sheet with an N-terminal helix - and the intersubunitbeta-sheet formation involving beta-strands 4 and 5 ofneighbouring subunits is similar to that seen in the crystalstructures of two dimeric complexes of human Sm proteinsrecently solved by Nagai and coworkers1. This suggests aclose evolutionary relationship, however, based on thedimeric complexes these authors have proposed a seven-membered, hetero-oligomeric ring structure for the humansnRNP core domain. Interestingly, preliminary resultsfrom the X-ray analysis of the PA-Sm1 protein alsoindicate a heptameric arrangement.  Solution studies andimmunoprecipitation experiments are under way to furthercharacterize the archaeal Sm-related proteins and toidentify their RNA targets.[1] Kambach, C., Walke, S., Young, R., Avis, J.M., de la Fortelle, E.,Raker, V., Lührmann, R., Li, J. & Nagai, K. (1999) Crystal structures oftwo Sm protein complexes and their implications for the assembly of thespliceosomal sn RNPs. Cell 96, 375-387.

Crystallographic dissection of the spliceosomal snRNP structures

http://discovery.ucl.ac.uk/1437831/1/acta%20cryst%20a.pdf

Crystallographic dissection of the spliceosomal snRNP structures

Abstract

Similar works

Full text

Available Versions

UCL Discovery