U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementaritiesm to 28S rRNA

Abstract

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

“…Genetic depletion of Nop58p+ A: Schematic representation of the structure of the GAL::nop58 allele+ B: Growth of the GAL::nop58 (circles) and NOP58 (squares) strains following transfer to glucose medium+ Cell density was measured at regular intervals and the cultures were periodically diluted to be continuously kept in exponential growth+ The results are presented on an exponential scale with the OD values corrected for the dilution factor+ C: Northern hybridization of the NOP58 mRNA in a GAL::nop58 strain+ RNA was extracted from NOP58 and GAL::nop58 strains following growth on permissive rsg medium (0-h lanes) and at intervals following transfer to glucose medium (6-24-h lanes) and separated on a 1+2% agarose gel containing formaldehyde+ ization using specific oligonucleotide probes or antisense RNA transcripts (see Materials and Methods)+ Strong depletion was observed for all tested box CϩD snoRNAs: U3, U14, U18, snR4, snR190 ( Among the box CϩD snoRNAs tested, some variation in sensitivity to the depletion of Nop58p was observed, with higher residual levels of U3 and U24 than other species+ For both of these snoRNAs low levels of longer forms were detected on depletion of Nop58p (Figs+ 4A and 5)+ These extended species were investigated in more detail for U24+ Higher resolution Northerns showed that both shorter and longer forms of U24 were accumulated (Fig+ 5B)+ Primer extension from an internal U24 oligonucleotide revealed that the 59 end of U24 is unaltered in the Nop58p depleted strain (Fig+ 5C)+ RNase protection was used to map the 39 ends of U24 (Fig+ 5D)+ An antisense transcript overlapping the 39 end of U24 was annealed to total RNA+ The RNA hybrids formed were digested with RNase A ϩ T1 and the protected fragments were resolved on a polyacrylamide gel (see Materials and Methods)+ The signal detected in the wild-type NOP58 strain correspond to the position of the authentic 39 end of U24+ In the GAL::nop58 strain, a protected fragment extended by 5 or 6 nt is detected and accumulates over the time course of depletion (Fig+ 5D)+ This would be in good agreement with the gel mobility of the major extended RNA species seen by Northern hybridization+ Why the band corresponding to the size of the mature U24 is not lost from the RNase protection during Nop58p depletion is unclear; we assume this to be an artifact due to the structure of the snoRNA or antisense RNA transcript+ Yeast U24 is encoded in the intron of the BEL1 gene (Qu et al+, 1995) and is produced from the debranched intron-lariat by exonuclease activities (Ooi et al+, 1998; FIGURE 4. The box CϩD snoRNAs are specifically depleted in GAL::nop58 strains+ RNA was extracted from NOP58 and GAL::nop58 strains following growth on permissive rsg medium (0-h lanes) and at intervals following transfer to glucose medium (6-24-h lanes), separated on a 8% polyacrylamide gel and analyzed by Northern hybridization+ A: Probes specific for box CϩD snoRNAs+ B: Probes specific for box HϩACA snoRNAs+ C: Probe to the RNase MRP RNA+ Petfalski et al+, 1998)+ We conclude that depletion of Nop58p interferes with normal 39 processing of U24+…”

“…Comparison of different box CϩD snoRNAs revealed some variation in the residual levels on depletion of Nop58p+ For U3, a longer species accumulated, clearly showing an effect on synthesis of the snoRNA+ The 59 end of mature U3 corresponds to the tri-methyl guanosine cap of the primary transcript, and the extended form is therefore very likely to be 39 extended+ In vertebrates and yeast, spliceosomal snRNAs are processed from precursors with 39 extensions (Madore et al+, 1984a(Madore et al+, , 1984bYuo et al+, 1985;Chanfreau et al+, 1997;Abou Elela & Ares, 1998), and U3 may be similarly processed+ Longer forms of U24 were also accumulated, together with low levels of shorter forms+ The 59 end of U24 was unaffected by depletion of Nop58p whereas RNase protection experiments detected species 39 extended by 5-6 nt, showing these changes to be due to alterations in 39 processing+ Yeast U24 is encoded in the intron of the BEL1 gene (Qu et al+, 1995) and the snoRNA is obligatorily synthesized from the intron following debranching of the intron lariat; very low levels of mature U24 are synthesized in a dbr1-⌬ strain that lacks debranching activity (Ooi et al+, 1998;Petfalski et al+, 1998)+ This strongly indicates that both ends of U24 are synthesized by exonucleolytic activities and, indeed, the 59 r 39 exonucleases Rat1p and Xrn1p were identified as the activities responsible for the 59 processing (Petfalski et al+, 1998)+ In vertebrates, the 59 and 39 ends of all tested intronencoded snoRNAs are synthesized by exonucleases (Cecconi et al+, 1995;Kiss & Filipowicz, 1995;Caffarelli et al+, 1996;Xia et al+, 1997)+…”

“…The box CϩD sequences, together with a stem structure that normally brings them together in the snoRNA secondary structure are the only elements essential for the synthesis and stability of this class of snoRNA, and their presence is the only feature that is clearly conserved among the box CϩD snoRNAs (Baserga et al+, 1991;Huang et al+, 1992;Caffarelli et al+, 1996;Watkins et al+, 1996;Xia et al+, 1997)+ A simple model for the involvement of Nop58p in snoRNA stability is through protection from the exonucleolytic activities that normally generate the mature ends+ If so, the alteration in the 39 end of U24 on depletion of Nop58p suggests that this interaction might be via binding to the conserved box D at the 39 end of the snoRNAs+ Curiously, inspection of the 39 end of U24 (CUGAUGAAU OH ) (Qu et al+, 1995) reveals the presence of a consensus box C motif (UGAUGA), partially overlapping the box D element (CUGA)+ We speculate that on depletion of Nop58p, the putative box C binding protein(s), which normally binds to the 59 end of the snoRNA, also binds to the cryptic 39 box C element+ This might confer extra protection to U24 and explain why this species is more resistant to Nop58p depletion+ During depletion of Nop58p, Nop1p was reported to be delocalized to the nucleoplasm and cytoplasm (Wu et al+, 1998)+ In the conditions used (up to 12 h in glucose medium) the snoRNAs were presumably strongly depleted+ This indicates that Nop1p does not localize to the nucleolus on its own, but rather is localized to and/or anchored to the nucleolus in association with snoRNPs+ The association of the box CϩD snoRNAs with the nucleolus does not require complementarity to the pre-rRNA (Lange et al+, 1998b)+ Instead, both snoRNA stability and nucleolar targeting require the conserved box CϩD elements and the terminal stem (Lange et al+, 1998a(Lange et al+, , 1998cSamarsky et al+, 1998)+ It seems probable that Nop58p stabilizes the snoRNAs via binding to one or both of these sequence elements and is, therefore, a good candidate to provide the nucleolar targeting signals+ Testing of this hypothesis will require the separation of the role of Nop58p in snoRNA stability from its putative targeting function+…”

A Novel Brain-specific Box C/D Small Nucleolar RNA Processed from Tandemly Repeated Introns of a Noncoding RNA Gene in Rats

“…3D and data not shown). In contrast, known ubiquitous intronic C/D box snoRNAs, whether they are encoded in protein genes or in noncoding RNAs, are conserved over a much larger span of vertebrate phylogeny (9,37,39,43,46). The three previously reported brain-specific C/D box snoRNAs also seem substantially more ancient than RBII-36, since their origin must predate the primates/rodents split during mammalian evolution (17).…”

“…RBII-36 snoRNA Is Only Detected in Rats-Most box C/D snoRNAs have homologues in distant vertebrate species that can be detected by Northern cross-hybridizations using oligonucleotide probes directed to their conserved antisense element (9,43,46). Given that the 3Ј half of RBII-36 snoRNA sequence is preferentially conserved among the different tandemly repeated units (Fig.…”