Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing

Abstract

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5Ј trimethyl guanosine (TMG) cap, ten 2Ј-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 5Ј end of U2. We further show that 2Ј-O-methylation and pseudouridylation activities reside in the nucleus and that the 5Ј 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.

Pseudouridines in spliceosomal snRNAs

“…1) (Yu et al, 1998). This stood in stark contrast to the results when unmodified U2 was used, where no higher-order splicing complexes were detected after a 3.5 h reconstitution.…”

“…In 1998, Yu et al developed a Xenopus oocyte reconstitution system to study U2 snRNA modification (Yu et al, 1998). In their work, an antisense U2 DNA oligonucleotide was injected into Xenopus oocytes to hybridize with endogenous U2 snRNA, thereby triggering endogenous RNase H activity that degraded the RNA strand (U2) of the RNA-DNA hybrid.…”

“…U2 modifications occurred slowly in some regions (no modification or very light modification observed within 3.5 h), and thus the function (or lack thereof) of injected U2 in snRNP assembly and pre-mRNA splicing reflected the importance of modifications in these particular regions. Using this method, Yu et al identified functionally important modified nucleotides within the first 27 nucleotides of U2 snRNA, including three pseudouridines (Yu et al, 1998) (Fig. 2 and 3).…”

“…About 15 years ago, though, several laboratories began to turn their attention to the mechanisms and functions of these modifications in spliceosomal snRNAs and rRNAs (Bachellerie et al, 1995;Cavaillé et al, 1996;KissLászló et al, 1996;Tycowski et al, 1996;Ganot et al, 1997;Ni et al, 1997;Smith and Steitz, 1997;Tycowski et al, 1998;Yu et al, 1998;Lowe and Eddy, 1999). Multiple effective assays and systems have since been developed for RNA modification research [reviewed in (Wu et al, 2011b)].…”

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

“…These 2′-O-methyl sugar-and pseudouridine-base modifications of the U snRNPs (Darzacq et al 2002;Jady et al 2003;Verheggen et al 2002) are guided by small CB-specific RNAs (scaRNAs) and are crucial for proper pre-mRNA splicing in vivo (Yu et al 1998). …”