Structure and function of an RNase H domain at the heart of the spliceosome.
Precursor-messenger RNA (pre-mRNA) splicing encompasses two sequential transesterification reactions in distinct active sites of the spliceosome that are transiently established by the interplay of small nuclear (sn) RNAs and spliceosomal proteins. Protein Prp8 is an active site component but the molecular mechanisms, by which it might facilitate splicing catalysis, are unknown. We have determined crystal structures of corresponding portions of yeast and human Prp8 that interact with functional regions of the pre-mRNA, revealing a phylogenetically conserved RNase H fold, augmented by Prp8-specific elements. Comparisons to RNase H-substrate complexes suggested how an RNA encompassing a 5'-splice site (SS) could bind relative to Prp8 residues, which on mutation, suppress splice defects in pre-mRNAs and snRNAs. A truncated RNase H-like active centre lies next to a known contact region of the 5'SS and directed mutagenesis confirmed that this centre is a functional hotspot. These data suggest that Prp8 employs an RNase H domain to help assemble and stabilize the spliceosomal catalytic core, coordinate the activities of other splicing factors and possibly participate in chemical catalysis of splicing.
Abteilung Zelluläre Biochemie, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.
Vladimir
V
Pena
Alexey
A
Rozov
Patrizia
P
Fabrizio
Reinhard
R
Lührmann
Markus C
MC
Wahl
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011743:Pyrimidines; D012322:RNA Precursors / Q000378:metabolism; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D016914:Ribonuclease H / Q000737:chemistry*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017381:Spliceosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D050505:Mutant Proteins; C077018:PRP8 protein, S cerevisiae; C115374:PRPF8 protein, human; D010455:Peptides; D011743:Pyrimidines; D012322:RNA Precursors; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D016914:Ribonuclease H; C030986:pyrimidine