Elements essential for accumulation and function of small nucleolar RNAs directing site-specific pseudouridylation of ribosomal RNAs.

During site-specific pseudouridylation of eukaryotic rRNAs, selection of correct substrate uridines for isomerization into pseudouridine is directed by small nucleolar RNAs (snoRNAs). The pseudouridylation guide snoRNAs share a common 'hairpin-hinge- hairpin-tail' secondary structure and two conserved sequence motifs, the H and ACA boxes, located in the single-stranded hinge and tail regions, respectively. In the 5'- and/or 3'-terminal hairpin, an internal loop structure, the pseudouridylation pocket, selects the target uridine through formation of base-pairing interactions with rRNAs. Here, essential elements for accumulation and function of rRNA pseudouridylation guide snoRNAs have been analysed by expressing various mutant yeast snR5, snR36 and human U65 snoRNAs in yeast cells. We demonstrate that the H and ACA boxes that are required for formation of the correct 5' and 3' ends of the snoRNA, respectively, are also essential for the pseudouridylation reaction directed by both the 5'- and 3'-terminal pseudouridylation pockets. Similarly, RNA helices flanking the two pseudouridylation pockets are equally essential for pseudouridylation reactions mediated by either the 5' or 3' hairpin structure, indicating that the two hairpin domains function in a highly co-operative manner. Finally, we demonstrate that by manipulating the rRNA recognition motifs of pseudouridylation guide snoRNAs, novel pseudouridylation sites can be generated in yeast rRNAs.     

In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.

It was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions.     

U3 snoRNA genes with and without intron in the Kluyveromyces genus: yeasts can accommodate great variations of the U3 snoRNA 3'-terminal domain.

The U3 snoRNA coding sequences from the genomic DNAs of Kluyveromyces delphensis and four variants of the Kluyveromyces marxianus species were cloned by PCR amplification. Nucleotide sequence analysis of the amplification products revealed a unique U3 snoRNA gene sequence in all the strains studied, except for K. marxianus var. fragilis. The K. marxianus U3 genes were intronless, whereas an intron similar to those of the Saccharomyces cerevisiae U3 genes was found in K. delphensis. Hence, U3 genes with and without intron are found in yeasts of the Saccharomycetoideae subfamily. The secondary structure of the K. delphensis pre-U3 snoRNA and of the K. marxianus mature snoRNAs were studied experimentally. They revealed a strong conservation in yeasts of (1) the architecture of U3 snoRNA introns, (2) the 5'-terminal domain of the mature snoRNA, and (3) the protein-anchoring regions of the U3 snoRNA 3' domain. In contrast, stem-loop structures 2, 3, and 4 of the 3' domain showed great variations in size, sequence, and structure. Using a genetic test, we show that, in spite of these variations, the Kluyveromyces U3 snoRNAs are functional in S. cerevisiae. We also show that S. cerevisiae U3A snoRNAs lacking the stem-loop structure 2 or 4 are functional. Hence, U3 snoRNA function can accommodate great variations of the RNA 3'-terminal domain.     

Processing of intron-encoded box C/D small nucleolar RNAs lacking a 5',3'-terminal stem structure

The C and D box-containing (box C/D) small nucleolar RNAs (snoRNAs) function in the nucleolytic processing and 2'-O-methylation of precursor rRNA. In vertebrates, most box C/D snoRNAs are processed from debranched pre-mRNA introns by exonucleolytic activities. Elements directing accurate snoRNA excision are located within the snoRNA itself; they comprise the conserved C and D boxes and an adjoining 5',3'-terminal stem. Although the terminal stem has been demonstrated to be essential for snoRNA accumulation, many snoRNAs lack a terminal helix. To identify the cis-acting elements supporting the accumulation of intron-encoded box C/D snoRNAs devoid of a terminal stem, we have investigated the in vivo processing of the human U46 snoRNA and an artificial snoRNA from the human beta-globin pre-mRNA. We demonstrate that internal and/or external stem structures located within the snoRNA or in the intronic flanking sequences support the accumulation of mammalian box C/D snoRNAs lacking a canonical terminal stem. In the intronic precursor RNA, transiently formed external and/or stable internal base-pairing interactions fold the C and D boxes together and therefore facilitate the binding of snoRNP proteins. Since the external intronic stems are degraded during snoRNA processing, we propose that the C and D boxes alone can provide metabolic stability for the mature snoRNA.     

Purification,cloning, and characterization of XendoU,a novel endoribonuclease involved in processing of intron-encoded small nucleolar RNAs in Xenopus laevis

Here we report the purification, from Xenopus laevis oocyte nuclear extracts, of a new endoribonuclease, XendoU, that is involved in the processing of the intron-encoded box C/D U16 small nucleolar RNA (snoRNA) from its host pre-mRNA. Such an activity has never been reported before and has several uncommon features that make it quite a novel enzyme: it is poly(U)-specific, it requires Mn(2+) ions, and it produces molecules with 2'-3'-cyclic phosphate termini. Even if XendoU cleaves U-stretches, it displays some preferential cleavage on snoRNA precursor molecules. XendoU also participates in the biosynthesis of another intron-encoded snoRNA, U86, which is contained in the NOP56 gene of Xenopus laevis. A common feature of these snoRNAs is that their production is alternative to that of the mRNA, suggesting an important regulatory role for all the factors involved in the processing reaction.     

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability.