"RNomics: an Experimental Approach that Identifies 201 Candidates for Novel, Small, Non-Messenger RNAs in Mouse".

Institute of Experimental Pathology/Molecular Neurobiology, ZMBE, 48149 Münster, Germany,
² Max-Planck-Institute of Molecular Genetics, 14195 Berlin-Dahlem, Germany and
⁵ Laboratoire de Biologie Moléculaire Eucaryote du CNRS, Université Paul-Sabatier, 31062 Toulouse, France

³ Present address: GPC Biotech AG, 82152 Plannegg-Martinsried, Germany
⁴ Present address: Department of Clinical Pharmacology, RCSI, Dublin 2, Ireland
¹ Corresponding authors e-mail:  huttenh@uni-muenster.de, brosius@uni-muenster.de   or bachel@ibcg.biotoul.fr

In mouse brain cDNA libraries generated from small RNA moleculeswe have identified a total of 201 different expressed RNA sequencespotentially 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.
Supplementary Data at:   http://exppc01.uni-muenster.de/expath/snmrnas.htm

A major goal of the joint international efforts of the Human Genome Project is the sequence, identification, structure, regulation and function of all 30,000-40,000 genes and their products. To facilitate functional analysis of the encoded gene products, this endeavor has been extended to model organisms from bacteria to mouse. Furthermore, expressed sequence tags (ESTs) have been employed to catalogue all mRNAs and recent efforts to generate their full-length sequences provide essential tools to study post-transcriptional processing of transcripts including alternative splicing, identification of protein coding genes and functional analysis. In contrast, not many experimental efforts address the class of small non-messenger RNAs (snmRNAs; Kiss-Laszlo et al, 1996; Olivias et al, 1997). These molecules do not encode proteins, but have cellular functions on their own or in complex with proteins that are bound to the RNA and thus form ribonucleoprotein complexes (RNPs). Such RNPs, found in cellular compartments as diverse as the nucleolus or dendritic processes of nerve cells (Tiedge et al, 1993; Pederson, 1998), exhibit a surprisingly diverse range of functions. However, the biological role of some of them remains elusive. Moreover, most systematic genomic searches are biased against their detection, and comprehensive identification by computational analysis of the genomic sequence of any organism remains an unsolved problem (Eddy, 1999). Hundreds of genes and their RNA products may thus remain undetected. Their functions, interactions in cellular circuits and roles in disease would remain unknown and our understanding of the functioning of a cell would be incomplete. Therefore, we set out to identify directly snmRNAs and their genes in the human genome and those of various model organisms.

Here we describe our experimental approach to the discovery of novel snmRNAs in mouse. This EST-like approach has been tailored for the detection of small RNAs [starting with material usually discarded: small total RNA in the size range ~50-500 nucleotides (nt)]. The resulting sequences have been termed expressed RNA sequences (ERNS). In this study, we present the first unbiased look at the small RNA population in a mammalian cell. Thus far, we have identified ~200 candidates for novel snmRNA species via ERNS. More than half of them correspond to new members of the two expanding subclasses of small nucleolar RNAs (snoRNAs) that guide RNA ribose methylation and pseudouridylation. Interestingly, while the vast majority of previously known members of these two snoRNA classes direct the modification of rRNA, several of the novel members are able to guide modification of spliceosomal small nuclear RNAs (snRNAs). Moeover, an unexpectedly large number of them remain without identified RNA targets. Finally, some of them are not ubiquitously expressed, as expected for rRNA or snRNA modification guides, raising the possibility of tissue-specific targets, presumably mRNAs.

This study represents the first unbiased look at the population of snmRNA species in a mammalian cell, providing the basis for a comprehensive understanding of genomic, cellular and organismal function. By our experimental approach, we could identify a large set of  novel snoRNAs of the C/D or H/ACA box type guiding ribose methylation or pseudouridylation not only in rRNA, as expected, but also in snRNAs. For the first time, we report the detection of guide snoRNAs directing ribose methylations in U2 and U4 snRNAs, as well as snoRNA guides for pseudouridylations in U2 and U6 snRNAs. In addition, we identified a surprisingly large number of snoRNA species from both classes without the potential to target rRNAs or snRNAs, as deduced from their lack of appropriate complementarity. Especially intriguing was the identification of several brain-specific snmRNAs, all of which belong to the snoRNA type. This might lead to further studies to identify snoRNAs expressed tissue specifically in tissues other than brain. One of the brain-specific snoRNAs  (MBII-52) has been suggested to target serotonin receptor 5-HT_2C hnRNA or mRNA, which in turn is expressed specifically in brain (Cavaille et al, 2000). This may be indicative of a novel function of snoRNAs, namely the regulation of gene expression by binding to and/or modifying mRNAs or their hnRNA precursors via their antisense elements. At this stage, it is difficult to speculate about the function of potential snmRNAs of the non-snoRNA type. As demonstrated, some of these novel species are derived from hnRNAs or mRNAs and might therefore correspond to degradation products of larger transcripts. Alternatively, they could regulate the expression of mRNAs by as yet unknown mechanisms, especially when located within their 5' or 3' UTRs. Their detection sets the stage for direct experimental testing of these hypotheses.
Supplementary Data at:   http://exppc01.uni-muenster.de/expath/snmrnas.htm

Identification of Novel RNA Species:

We prepared total RNA from mouse brains by the TRIzol method (Gibco-BRL). Total RNA was subsequently fractionated on a denaturing polyacrylamide gel (7 M urea, 1X TBE buffer). RNAs in the size range ~50 to ~110 (Fraction II) or ~110 to ~500 (Fraction I) were excised from the gel, passively eluted and ethanol precipitated. Subsequently, 5 ug of RNA were tailed with CTP using poly(A) polymerase, as described by DeChiara and Brosius (1987). RNAs were reverse transcribed into cDNAs using primer GIBCO1 (see supplementary data), and cloned into pSPORT 1 vector employing the GIBCO Superscript^TM system (Gibco-BRL). cDNAs were amplified by PCR using primers FSP and RSP (see supplementary data).
PCR products were spotted by robots in high density arrays onto filters by the method of Schmitt et al. (1999), performed at the Resource Center of the German Human Genome Project (Berlin, Germany).

Filter Hybridization and Isolation of Clones:

For exclusion of the most abundant, known, small RNA species, we end-labelled oligonucleotides (see Supplementary data) derived from these sequences with [³²P]ATP and T4 polynucleotide kinase, and hybridized oligonucleotides to DNA arrays spotted on filters (see above). We performed hybridization in 0.5 M sodium phosphate pH 7.2, 7% SDS, 1 mM EDTA at 53^oC for 12 h. We washed filters twice at room temperature for 15 min in 40 mM sodium phosphate buffer pH 7.2, 0.1% SDS, exposed fibers to a phosphoimaging screen and analyzed filters by computer-aided determination of hybridization signals (Maier et al., 1994).

Accession Numbers of Sequences:

The sequence data have been submitted to the DDBJ/EMBL/GenBank databases under the accession numbers AF357317-AF357517.

Supplementary Data:

For additional methods see Supplementary data available at The EMBO Journal Online. These data will also be available and periodically updated at our web page:
 http://exppc01.uni-muenster.de/expath/snmrnas.htm

1. "Imprinted Expression of  Small Nucleolar RNAs in Brain: Time for RNomics".

2. "Identification of  Brain-Specific and Imprinted Small Nucleolar RNA Genes Exhibiting an Unusual Genomic Organization".

3. "Activation of DNA Transcription within Repressed Chromatin by Nuclear RNA Species".