Characterization of the Intron-Encoded U19 RNA, a New Mammalian Small Nucleolar RNA That Is Not Associated with Fibrillarin

Abstract

We have characterized a new member (U19) of a group of mammalian small nucleolar RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date. Human U19 RNA is 200 nucleotides long and possesses 5-monophosphate and 3-hydroxyl termini. It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs. Human and mouse RNAs are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA. In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts. During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of ϳ65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.The 18S, 5.8S, and 25/28S rRNAs are synthesized in the nucleolus as a single precursor RNA (pre-rRNA) which contains additional sequences that are discarded during RNA maturation. Processing of the pre-rRNA is a complex process involving many RNA intermediates and cleavage events, which frequently follow alternative pathways. The substrate for processing is a large ribonucleoprotein structure containing tens of ribosomal proteins and nucleolar accessory factors associated with nascent pre-rRNA (reviewed in references 10, 53, 60, and 61).Nucleoli contain a large number of metabolically stable 60-to 260-nucleotide (nt)-long RNA species, referred to as small nucleolar RNAs (snoRNAs) (reviewed in references 13, 14, 37, and 53). Some of these RNAs, e.g., U3, U8, and U13, are transcribed from independent transcription units and contain 5Ј-terminal trimethylguanosine caps. Most snoRNAs, however, are processed from introns of pre-mRNAs (32; reviewed in references 13, 37, and 52) and contain a monophosphate at the 5Ј end (24,42,57), consistent with their processing from longer precursor RNAs.Several snoRNAs have been demonstrated to participate in the processing of pre-rRNA. The U3 snoRNA is involved in the earliest cleavage event, which occurs in the 5Ј external transcribed spacer (19,21,41), and its depletion impairs the accumulation of mature 18S rRNA in the yeast Saccharomyces cerevisiae and vertebrates (19,50). snoRNAs U14, snR10, and snR30 in S. cerevisiae (33, 39, 54) and U22 in Xenopus laevis (58) are also essential for 18S rRNA processing, while 7-2/ MRP RNA and U8 snoRNA are involved in a maturation of 5.8S rRNA in S. cerevisiae (9,35,51) and 28S rRNA in X. laevis (43), respectively. On the basis of their association with prerRNAs or with nascent ribosomal particles, other snoRNAs may also participate in pre-rRNA processing or other aspects of ribosome biogenesis in the nucleolus (1,2,14,37,53,60).The snoRNAs are complexed with proteins, forming small ribonucleopro...

Human H/ACA Small Nucleolar RNPs and Telomerase Share Evolutionarily Conserved Proteins NHP2 and NOP10

“…Individual RNA species were identified by an RNase A/T1 protection assay as described previously (26) using antisense RNA probes complementary to U17 (37), U3 (35), U19 (38), U13 (36), and hTR (15). Probes were labeled with [␣-32 P]UTP (800 Ci/mmol; NEN).…”

Dynamic association and localization of human H/ACA RNP proteins

“…GFP-NAF1 with the mutated NLS (pTM184) was generated based on pNK16 by pairwise replacement of two lysines by alanines using the QuickChange II kit (Stratagene) according to the manufacturer's instructions. The probes for RNase protection assays of rat and human E3 snoRNA (Darzacq et al 2006) and for U19 (Kiss et al 1996) were as published.…”

A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast

“…5). Figure 5 shows that, in these conditions, the presence of snR191 gives a small yet reproducible growth advantage to the cells: in six independent cultures, the wild type represented ∼50% (Kiss et al 1996). Note that the S. bayanus and K. marxianus sequences are issued from the Génolevure project (Souciet et al 2000) and thus correspond to single reads that are prone to contain some errors or ambiguities (e.g., M stands for A or C).…”

“…One known human snoRNA, hU19 (Kiss et al 1996), has been previously proposed, on the basis of its structure and sequence, to specify these two highly conserved pseudouridines in the human LSU rRNA (Bortolin and Kiss 1998). The alignment of the hU19 snoRNA sequence with yeast snR191 is shown in Figure 2A and the comparison of the potential RNA secondary structures for these RNAs is shown in Figure 2B,E.…”

“…Among all snR191 potential secondary structures predicted using the MFOLD program (Walter et al 1994), the ones that are conserved between S. cerevisiae (B), S. bayanus (C), and K. marxianus (D) are shown. (E) Secondary structure of human hU19 RNA as determined in Kiss et al (1996). The potential guide sequences are boxed and labeled AAЈ and BBЈ in the S. cerevisiae structure in B.…”