A small nucleolar guide RNA functions both in 2'-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA

In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2'-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2'-O-methylation experiments provide evidence that the U85 snoRNA directs 2'-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2'-O-methylation.     

Guided tours: from precursor snoRNA to functional snoRNP.

Small nucleolar RNAs (snoRNAs) use base pairing to guide modification of conserved nucleotides in functionally important regions of ribosomal RNA. The box C/D snoRNAs direct 2'-O-methylation and the box H/ACA snoRNAs direct pseudouridylation. Each snoRNA interacts with proteins, many of them newly identified. Progress in understanding how snoRNA sequences are stored within genomes, liberated from precursor molecules and targeted to the nucleolus has begun to elucidate each step in the biogenesis of these critical contributors to ribosome formation.     

Probing RNA in vivo with methylation guide small nucleolar RNAs

Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2'-O-methylated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex. Sites to be modified are selected by a long (>10-nt) antisense guide sequence in the snoRNA and a distance measurement from a box D or D' element that follows the snoRNA guide sequence. Modification of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D'. Methylation can be targeted to novel sites by expressing a snoRNA with a new guide sequence. In some cases methylation impairs the growth rate of the cell, indicating that a functionally important nucleotide has been altered. With a view to harnessing snoRNA-directed methylation for functional mapping, we have developed a method for constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest. The strategy and procedures are described here, and preliminary results are presented that show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center.     

Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2'-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs

In eukaryotic cells, the site-specific 2′-O-ribose methy-lation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2′-O-methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10–21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2′-O-methylation guide snoRNAs. However, in contrast to all of the known 2′-O-methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2′-O-methy-lated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2′-O-methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.     

An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes

U2 is the most extensively modified of all spliceosomal snRNAs. We previously showed that at least some of the internally modified nucleotides in U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing. Recent work from several laboratories suggests that nuclear guide RNAs facilitate U2 snRNA internal modification, including pseudouridylation and 2'-O-methylation. Here, we present a novel approach to identifying guide RNAs for U2 pseudouridylation. Several Xenopus oocyte nuclear RNAs were affinity selected with U2 snRNA substituted with 5-fluorouridine, a pseudouridylation inhibitor that sequesters pseudouridylases. One of these RNAs was sequenced and found to be a novel RNA of 134 nt. This small RNA contains an H/ACA motif and folds into a typical H/ACA RNA structure, and its authenticity as an H/ACA RNA was confirmed by immunoprecipitation analysis. The RNA contains two guide sequences for pseudouridylation (psi) of U2 snRNA at positions 34 and 44 in the branch-site recognition region, and we demonstrate that this RNA indeed guides the formation of psi34 and psi44 in U2 using a Xenopus oocyte reconstitution system. Therefore, this novel RNA was designated pugU2-34/44, for pseudouridylation guide for U2 snRNA U34 and U44. Intranuclear localization analyses indicate that pugU2-34/44 resides within the nucleoplasm rather than nucleoli or Cajal bodies where other guide RNAs have been localized. Our results clarify the mechanism of U2 snRNA pseudouridylation in Xenopus oocytes, and have interesting implications with regard to the intranuclear localization of U2 snRNA pseudouridylation.     

Targeted pre-mRNA modification for gene silencing and regulation

Most eukaryotic box C/D small nucleolar (sno) or Cajal body-specific RNAs guide base pairing with target RNAs and direct site-specific 2'-O-methylation. We designed an artificial C/D RNA to target the branch point adenosine of ACT1 pre-mRNA to block its splicing. Saccharomyces cerevisiae expressing this guide RNA gene controlled by a GAL1 promoter grew normally on dextrose but not on galactose medium. The pre-mRNA was specifically 2'-O-methylated, prohibiting maturation of ACT1 mRNA. Targeting other adenosines in this region while maintaining almost identical complementarity did not affect ACT1 mRNA level or cell growth, suggesting that targeting the branch-point adenosine was truly 2'-O-methylation-specific rather than an antisense effect; moreover, only the 3'-most branch site adenosine served as the branch point. We targeted other essential intron-containing genes, and observed a similar phenotype. We demonstrated that a Box C/D RNA can guide modification at the pre-mRNA branch point, thus silencing its expression and inducing cell death.     

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.     

A common maturation pathway for small nucleolar RNAs.

We have shown that precursors of U3, U8 and U14 small nucleolar RNAs (snoRNAs) are not exported to the cytoplasm after injection into Xenopus oocyte nuclei but are selectively retained and matured in the nucleus, where they function in pre-rRNA processing. Our results demonstrate that Box D, a conserved sequence element found in these and most other snoRNAs, plays a key role in their nuclear retention, 5' cap hypermethylation and stability. Retention of U3 and U8 RNAs in the nucleus is saturable and relies on one or more common factors. Hypermethylation of the 5' caps of U3 RNA occurs efficiently in oocyte nuclear extracts lacking nucleoli, suggesting that precursor snoRNAs are matured in the nucleoplasm before they are localized to the nucleolus. Surprisingly, m7G-capped precursors of spliceosomal small nuclear RNAs (snRNAs) such as pre-U1 and U2, can be hypermethylated in nuclei if the RNAs are complexed with Sm proteins. This raises the possibility that a single nuclear hypermethylase activity may act on both nucleolar and spliceosomal snRNPs.     

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.     

Biogenesis of small nucleolar ribonucleoproteins

Eukaryotic cells contain a very complex population of small nucleolar RNAs. They function, as small nucleolar ribonucleoproteins, in pre-ribosomal RNA processing reactions, and also guide methylation and pseudouridylation of ribosomal RNA, spliceosomal small nuclear RNAs, and possibly other cellular RNAs. Synthesis of small nucleolar RNAs frequently follows unusual strategies. Some newly discovered brain-specific small nucleolar RNAs of unknown function are encoded in introns of tandemly repeated units, expression of which is paternally imprinted. Recent studies of the protein components and factors participating in small nucleolar ribonucleoprotein assembly have revealed interesting connections with other classes of cellular ribonucleoproteins such as spliceosomal small nuclear ribonucleoproteins and telomerase. Cajal bodies emerge as nuclear structures important for the biogenesis and function of small nucleolar ribonucleoproteins.     

Sequential 2'-O-methylation of archaeal pre-tRNATrp nucleotides is guided by the intron-encoded but trans-acting box C/D ribonucleoprotein of pre-tRNA

Haloferax volcanii pre-tRNA(Trp) processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNA(Trp) in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNA(Trp) or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson-Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNA(Trp) added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intron-encoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pre-tRNA(Trp) intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing.     

Three new small nucleolar RNAs that are psoralen cross-linked in vivo to unique regions of pre-rRNA.

We have recently described three novel human small nucleolar RNA species with unique nucleotide sequences, which were named E1, E2, and E3. The present article describes specific psoralen photocross-linking in whole HeLa cells of E1, E2, and E3 RNAs to nucleolar pre-rRNA. These small RNAs were cross-linked to different sections of pre-rRNA. E1 RNA was cross-linked to two segments of nucleolar pre-rRNA; one was within residues 697 to 1163 of the 5' external transcribed spacer, and the other one was between nucleotides 664 and 1021 of the 18S rRNA sequence. E2 RNA was cross-linked to a region within residues 3282 to 3667 of the 28S rRNA sequence. E3 RNA was cross-linked to a sequence between positions 1021 and 1639 of the 18S rRNA sequence. Primer extension analysis located psoralen adducts in E1, E2, and E3 RNAs that were enriched in high-molecular-weight fractions of nucleolar RNA. Some of these psoralen adducts might be cross-links of E1, E2, and E3 RNAs to large nucleolar RNA. Antisense oligodeoxynucleotide-targeted RNase H digestion of nucleolar extracts revealed accessible segments in these three small RNAs. The accessible regions were within nucleotide positions 106 to 130 of E1 RNA, positions 24 to 48 and 42 to 66 of E2 RNA, and positions 7 to 16 and about 116 to 122 of E3 RNA. Some of the molecules of these small nucleolar RNAs sedimented as if associated with larger structures when both nondenatured RNA and a nucleolar extract were analyzed.