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Published online before print December 12, 2005, 10.1101/gr.4200206
Genome Res. 16:11-19, 2006
©2006 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/06 $5.00
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Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

Timothy Ravasi1,4,5, Harukazu Suzuki2,4, Ken C. Pang1,3,4, Shintaro Katayama2,4, Masaaki Furuno2,4,6, Rie Okunishi2, Shiro Fukuda2, Kelin Ru1, Martin C. Frith1,2, M. Milena Gongora1, Sean M. Grimmond1, David A. Hume1, Yoshihide Hayashizaki2 and John S. Mattick1,7

1 ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia 2 Laboratory for Genome Exploration Research Group, RIKEN Genomic Science Center, RIKEN Yokohama Institute, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan 3 T Cell Laboratory, Ludwig Institute for Cancer Research, Austin & Repatriation Medical Centre, Heidelberg VIC 3084, Australia

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific "transcriptional noise." Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Article published online ahead of print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.4200206.

4 These authors contributed equally to this work.

5 Present address: Department of Bioengineering, University of California-San Diego, La Jolla, CA 92093-0412, USA

6 Present address: Mouse Genome Informatics Consortium, The Jackson Laboratory, Bar Harbor, ME 04609, USA

7 Corresponding author.
E-mail j.mattick{at}imb.uq.edu.au; fax 61-7-3346-2111.

[Supplemental material is available online at www.genome.org. The microarray data from this study have been submitted to the Gene Expression Omnibus under accession nos. GSD275 and GSE3098 [NCBI GEO] .]


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