Three small nucleolar RNAs of unique nucleotide sequences.

Abstract

Three small RNA species were detected in human cells, and their cDNAs were synthesized and cloned. These RNAs are nucleolar, are 207, 154, and 135 nucleotides long, and are named El, E2, and E3, respectively, and their unique nucleotide sequences suggest that they may belong to an additional family of small nucleolar RNAs. The 5' ends of these three RNAs do not appear to have a trimethylguanosine cap or another type of cap. Apparent homologs of these three RNAs were detected in mouse, rabbit, and frog cells, suggesting their universal importance. They are housekeeping RNA species, since they are present in all rabbit tissues analyzed.The formation of cytoplasmic ribosomes in eukaryotic cells involves a complex series of synthesis, processing, assembly, and transport steps, many of which are poorly understood (reviewed recently in refs. 1 and 2). Some small nucleolar RNA (snoRNA) species, such as U3 RNA and yeast snR10 and U14 RNA, are required for rRNA precursor (pre-rRNA) processing (3-7). It has been difficult to study some of the low-abundance small nuclear RNAs in the absence of specific DNA or antibody probes. We have made cDNA probes for three snoRNAs and report some of the properties of these snoRNAs.t MATERIALS AND METHODS General Methods. The following procedures were done as described: preparation of nuclear and cytoplasmic fractions from HeLa cells (8); isolation ofnucleoli (9); isolation ofRNA (10); hybrid selection of RNA (8); 10%o polyacrylamide gel electrophoresis (8); electrophoresis of RNA in 1% agarose and 2.2 M formaldehyde (11); RNA sequencing by the dideoxynucleotide method, using HeLa cell nucleolar RNA, avian myeloblastosis virus reverse transcriptase, and sequence-specific, antisense radioactive primers (12); and immunoprecipitation assays (13). The recommendations of the corresponding manufacturers were followed for (i) hybridization with ZetaProbe membranes (Bio-Rad) and (ii) Radioactive Hybridization Probes. The El, E2, and E3 sequences in the corresponding cDNA clones, minus the tails that had been added for cDNA synthesis and cloning, were amplified by the polymerase chain reaction (14). The amplified products were then used as templates for extension of El-, E2-, and E3-specific, antisense 3' end primers with the Klenow fragment of DNA polymerase I in the presence of [a-32PldATP to generate the respective labeled, sequencespecific DNA probes. The radioactive DNA probe for Ul RNA was made by extension of a Ul-specific primer using as template the human Ul gene plasmid, linearized near the 5' end ofthe Ul gene. The radiolabeled DNA probe for U3 RNA was made by random primer labeling (15) using the rat U3 RNA sequence plasmid as template. RESULTSThere were some minor small RNA bands in HeLa cell nuclear RNA (bands labeled 1, 2, and 3 in lane 1 of Fig. 1) that could not be hybrid selected with any of the available DNA clones for small RNAs. RNA from these three sections of the gel was used to make three cDNA libraries. Clones from these libraries were used to probe Northern bl...

U17XS8, a small nucleolar RNA with a 12 nt complementarity to 18S rRNA and coded by a sequence repeated in the six introns ofXenopus laevisribosomal protein S8 gene

“…It is well known that a whole genome duplication occurred in Xenopus laevis about 30 million years ago (21), resulting in duplication followed by some limited divergence of all genes. That also r-protein genes are present in two copies, and in particular the gene for r-protein S8, has been previously demonstrated (22,23 U17a RNA (9,10). Comparison of these, strongly indicates a structure common to the seven of them ( Figure 4B shows the structures for U17Xs8 f and human U17a).…”

“…These examples are numerous enough to permit the conclusion that this gene organization is not coincidental but must have an important functional relevance. More intriguing is the case of U17 RNA, very recently described in HeLa cells (9,10). In fact this RNA, which is 69.3% homologous to Xenopus U17XS8 RNA, is encoded only in two introns present in the non coding region of the human gene for RCC 1, a protein involved in cell cycle regulation (9), while Ul7Xs8 RNA is encoded only in the introns of r-protein S8 genes.…”

“…A subgroup of these, the snoRNAs, is localized in the animal nucleolus and includes U3, U8, U13, U14 RNAs (31,28,6), the recently found U15, U16, U17 RNAs (7-9) and El (same as U 17), E2 and E3 RNAs (10). About (33)(34)(35)(36) and U8 RNA (37).…”

“…The name U 17 has been adopted in consideration of the sequence homology between our RNA and human U17 RNA (9), also called El RNA (10); however, because of the some differences in their structure and gene organization (see below), we added the superscript 'XS8' to identify the one encoded in the Xenopus S8a gene. The actual existence in the cell of this RNA species, at first observed only by Northern blot hybridization, has been proved by RNase mapping experiments.…”

“…We have adopted the name 'U 17' for the Xenopus S8a intron encoded snoRNA after consideration of the strong homology with the U17 RNA described in HeLa cells (9), also called El RNA by Ruff et al (10); however, because of some relevant differences in structure and properties, we identify the Xenopus RNA with a specifying superscript. 1 Itg of linear plasmid templates as described (15) using about 50 ,uCi of [(x-32p] CTP in a total CTP concentration of 10 mM, and gel purified. RNase mapping was performed according to Zinn et al.…”