RNPomics: defining the ncRNA transcriptome by cDNA library generation from ribonucleo-protein particles.
Up to 450,000 non-coding RNAs (ncRNAs) have been predicted to be transcribed from the human genome. However, it still has to be elucidated which of these transcripts represent functional ncRNAs. Since all functional ncRNAs in Eukarya form ribonucleo-protein particles (RNPs), we generated specialized cDNA libraries from size-fractionated RNPs and validated the presence of selected ncRNAs within RNPs by glycerol gradient centrifugation. As a proof of concept, we applied the RNP method to human Hela cells or total mouse brain, and subjected cDNA libraries, generated from the two model systems, to deep-sequencing. Bioinformatical analysis of cDNA sequences revealed several hundred ncRNP candidates. Thereby, ncRNAs candidates were mainly located in intergenic as well as intronic regions of the genome, with a significant overrepresentation of intron-derived ncRNA sequences. Additionally, a number of ncRNAs mapped to repetitive sequences. Thus, our RNP approach provides an efficient way to identify new functional small ncRNA candidates, involved in RNP formation.
Division of Genomics and RNomics, Innsbruck Biocentre, Innsbruck Medical University, Innsbruck and Institute of Theoretical Chemistry, University of Vienna, Vienna, Austria.
Mathieu
M
Rederstorff
Stephan H
SH
Bernhart
Andrea
A
Tanzer
Marek
M
Zywicki
Katrin
K
Perfler
Melanie
M
Lukasser
Ivo L
IL
Hofacker
Alexander
A
Hüttenhofer
D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D017124:Conserved Sequence; D005091:Exons; D020869:Gene Expression Profiling; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D051379:Mice; D022661:RNA, Untranslated / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000302:isolation & purification
D022661:RNA, Untranslated; D012261:Ribonucleoproteins