Abstract
BackgroundNoncoding RNAs (ncRNAs) play important roles in a variety of cellular processes. Characterizing the transcriptional activity of ncRNA promoters is therefore a critical step toward understanding the complex cellular roles of ncRNAs.ResultsHere we present an in vivo transcriptional analysis of three C. elegans ncRNA upstream motifs (UM1-3). Transcriptional activity of all three motifs has been demonstrated, and mutational analysis revealed differential contributions of different parts of each motif. We showed that upstream motif 1 (UM1) can drive the expression of green fluorescent protein (GFP), and utilized this for detailed analysis of temporal and spatial expression patterns of 5 SL2 RNAs. Upstream motifs 2 and 3 do not drive GFP expression, and termination at consecutive T runs suggests transcription by RNA polymerase III. The UM2 sequence resembles the tRNA promoter, and is actually embedded within its own short-lived, primary transcript. This is a structure which is also found at a few plant and yeast loci, and may indicate an evolutionarily very old dicistronic transcription pattern in which a tRNA serves as a promoter for an adjacent snoRNA.ConclusionThe study has demonstrated that the three upstream motifs UM1-3 have promoter activity. The UM1 sequence can drive expression of GFP, which allows for the use of UM1::GFP fusion constructs to study temporal-spatial expression patterns of UM1 ncRNA loci. The UM1 loci appear to act in concert with other upstream sequences, whereas the transcriptional activities of the UM2 and UM3 are confined to the motifs themselves.
Highlights
Noncoding RNAs play important roles in a variety of cellular processes
In order to analyse the transcriptional activities of the three common noncoding RNAs (ncRNAs) motifs, we made constructs containing varying amounts (~100 bp, ~300 bp and ~1 kb) of upstream sequences fused to the green fluorescent protein (GFP) open reading frame (ORF)
All the three upstream motifs are transcriptionally active Cloning of approximately 100 bp upstream sequence encompassing each of the three upstream motifs 1–3 in front of chimeric ncRNA::GFP reporter genes suggested that all three motifs have independent transcriptional activity
Summary
Characterizing the transcriptional activity of ncRNA promoters is a critical step toward understanding the complex cellular roles of ncRNAs. Genome wide analyses have in recent years revealed an increasing number of noncoding RNAs (ncRNAs) [1,2,3,4,5,6,7,8,9,10,11,12], the functional roles of these ncRNAs are mostly still unknown. Characterizing the transcriptional activity of the promoters of these loci could be a useful step towards revealing their functional roles. NAs and tRNAs not included) and a recent tiling microarray study suggests the existence of an additional 1200 short transcripts with unknown function (TUFs) [1]. Of the 1222 transcripts of unknown function (TUF)[1], UM1-3 are found at 76, 44 and 4 loci, respectively
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