RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

Abstract

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:

Fig. 1. Sequence analysis of 400 randomly chosen cDNA clones from mouse brain library Fraction I (derived from RNAs sized 500–110 nt) or Fraction II (derived from RNAs sized 110–50 nt), respectively. cDNA clones representing different RNA species or categories are shown as a percentage of total clones. The segment denoted snmRNAs identifies candidates for novel snmRNAs.

Fig. 2. Potential base-pairing interactions between novel mouse H/ACA snoRNAs and mouse rRNA (A) or snRNA (B). The snoRNA sequences in a 5′ to 3′ orientation are shown in the upper strands with the two H and ACA motifs boxed and the apical part of the snoRNA 5′ or 3′ hairpin domains schematized by a solid line. The two complementarities to the RNA target are always found within the large internal loop of one (or both) of their hairpin domains, invariably abutting its apex-proximal stem. Sequence coordinates in parentheses refer to snoRNAs for which the 5′ terminal sequence remains incomplete after database analysis. For other snoRNAs, nucleotides in the 5′ terminal sequence derived from databases are depicted as lower case letters. For rRNA or snRNA, sequence coordinates correspond to the respective human sequences to facilitate interpretation of data. Positions of pseudouridines are as reported by Maden (1990) for 18S rRNA, by Ofengand and Bakin (1997) for 28S rRNA and by Massenet et al. (1998) for mammalian snRNAs. Pseudouridines predicted to be directed by the snoRNA are denoted by Ψ, while other known pseudouridylation sites are indicated by U. In three cases, the uridine (indicated by an arrow) at the expected target position in the canonical, bipartite guide RNA duplex has not been reported to be pseudouridylated.