Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome.
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.
Ravasi
Timothy
T
ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia.
Suzuki
Harukazu
H
Pang
Ken C
KC
Katayama
Shintaro
S
Furuno
Masaaki
M
Okunishi
Rie
R
Fukuda
Shiro
S
Ru
Kelin
K
Frith
Martin C
MC
Gongora
M Milena
MM
Grimmond
Sean M
SM
Hume
David A
DA
Hayashizaki
Yoshihide
Y
Mattick
John S
JS
D000818:Animals; D015152:Blotting, Northern; D020869:Gene Expression Profiling; D015723:Gene Library; D016678:Genome / Q000235:genetics*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D009928:Organ Specificity / Q000235:genetics; D022661:RNA, Untranslated / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D022661:RNA, Untranslated