Guide RNAs with 5′ caps and novel box C/D snoRNA-like domains for modification of snRNAs in Metazoa
ABSTRACT Spliceosomal snRNAs and ribosomal RNAs in metazoans contain numerous modified residues that are functionally important. The most common modifications are site-specific 2'-O-methylation and pseudouridylation, both directed by small ribonucleoprotein particles. Each particle is composed of a short guide RNA and a set of several proteins. All previously characterized modification guide RNAs in metazoa are encoded in and processed from introns.
We have identified and characterized three novel guide RNAs for conserved 2'-O-methylation of U2, U4, and U12 snRNAs. Two guides, termed mgU2-25/61 and mgU12-22/U4-8, appear to be independently transcribed as judged by the presence of methylated guanosine caps at their 5' ends and upstream promoters similar to those of telomerase RNA. These guide RNAs are each composed of a canonical box C/D snoRNA and a novel box C/D snoRNA-like domain, where the C'/D' motif, rather than C/D, can be folded into a conserved kink-turn structure. The snoRNA-like domains are predicted to direct 2'-O-methylation of invariant G residues that occupy analogous positions in the U2 and U12 snRNA secondary structures. A third guide, mgU2-19/30 RNA, is composed of two canonical box C/D snoRNA domains encoded within a single intron.
This is the first description in metazoan cells of 5'-capped modification guide RNAs that appear to be independently transcribed. Since plant, yeast, and protozoan guide RNAs are mostly independently transcribed, the identification of such RNAs argues that ancestral metazoans possessed independently transcribed guide RNAs and only later, during the evolution of metazoan organisms, did the guide RNA genes shift to introns.
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- "Trimethyl-capped RNAs were immunoprecipitated using a strictly specific rabbit polyclonal anti-m3G (TMG) serum (Synaptic Systems) also referred to as R1131 (38,39). Ten microliters of serum was coupled to 50 μl of protein A-Sepharose beads saturated with 20 μg of both purified bovine serum albumin (BSA) and total yeast tRNA in NT2 buffer for 18 h at 4°C. "
ABSTRACT: Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.Nucleic Acids Research 07/2014; 42(13). DOI:10.1093/nar/gku580 · 9.11 Impact Factor
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- "The human mgU2-25/61 and mgU12-22/U4-8 box C/D scaRNAs, instead of being processed for pre-mRNA introns, are synthesized from independent genes by RNA Pol II and they carry methylated guanosine caps at their 5′ termini (29). While the 5′-terminal region of vertebrate mgU2–25/61 RNAs is remarkably rich in repeated GU and to less extent AU dinucleotides, the mgU12-22/U4-8 RNAs lack (GU) repeat sequences (Supplementary Figures S4 and S5). "
ABSTRACT: Post-transcriptional pseudouridylation and 2′-O-methylation of splicesomal small nuclear ribonucleic acids (snRNAs) is mediated by box H/ACA and box C/D small Cajal body (CB)-specific ribonucleoproteins (scaRNPs), respectively. The WD-repeat protein 79 (WDR79) has been proposed to interact with both classes of modification scaRNPs and target them into the CB. The box H/ACA scaRNAs carry the common CAB box motif (consensus, ugAG) that is required for both WDR79 binding and CB-specific accumulation. Thus far, no cis-acting CB-localization element has been reported for vertebrate box C/D scaRNAs. In this study, systematic mutational analysis of the human U90 and another newly identified box C/D scaRNA, mgU2-47, demonstrated that the CB-specific accumulation of vertebrate intron-encoded box C/D scaRNAs relies on GU- or UG-dominated dinucleotide repeat sequences which are predicted to form the terminal stem-loop of the RNA apical hairpin. While the loop nucleotides are unimportant, the adjacent terminal helix that is composed mostly of consecutive G.U and U.G wobble base-pairs is essential for CB-specific localization of box C/D scaRNAs. Co-immunoprecipitation experiments confirmed that the newly identified CB localization element, called the G.U/U.G wobble stem, is crucial for in vivo association of box C/D scaRNPs with WDR79.Nucleic Acids Research 04/2014; 42(10). DOI:10.1093/nar/gku287 · 9.11 Impact Factor
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- "The RNA sequencing results described above (Table 1) show that coilin immunocomplexes are greatly enriched for the box C/D scaRNAs 2 and 9. To examine if WRAP53 may be mediating this enrichment, IPs were conducted using lysate generated from HeLa cells transfected with control, coilin or WRAP53 siRNA, followed by RNA isolation, cDNA generation and qPCR using primers specific to scaRNA 2 and 9 (Fig. 3). Both scaRNA 2 and 9 are processed to yield smaller fragments that guide modifications on U2 snRNA (Tycowski et al., 2004). For scaRNA 2, mgU2-61 is generated while two guide RNAs are processed from scaRNA 9: mgU2-19 and mgU2-30. "
ABSTRACT: Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase.Biology Open 03/2014; 3(4). DOI:10.1242/bio.20147443 · 2.42 Impact Factor