Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.
Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.
Maroney
P A
PA
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Yu
Y T
YT
Jankowska
M
M
Nilsen
T W
TW
D000818:Animals; D017164:Ascaris lumbricoides / Q000235:genetics* / Q000378:metabolism; D001324:Autoantigens / Q000378:metabolism; D001483:Base Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D018510:RNA, Helminth / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D055517:snRNP Core Proteins
D001324:Autoantigens; D018510:RNA, Helminth; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins