Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing.
Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.
Department of Molecular Biophysics and Biochemistry, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
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Steitz
D000818:Animals; D001483:Base Sequence; D006151:Guanosine / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D009865:Oocytes; D011560:Pseudouridine / Q000031:analogs & derivatives; D012315:RNA Caps; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism; D014981:Xenopus
D012315:RNA Caps; D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D006151:Guanosine; D011560:Pseudouridine