Analysis of gene expression data in two C.elegans mutant strains: KP3293 tom-1(nu468) and KP3365 unc-43(n1186); hif-1(nu469). These results support the utility of microarray hybridizations to facilitate positional cloning. Keywords: other
The 3' untranslated region (3' UTR) constitutes a major site of post-transcriptional regulation of gene expression. Sequence elements in the 3' UTR interact with trans-acting regulators such as microRNAs that affect translation and stability. The overall aim is to use a 3'RACE cloning-sequencing stragety to identify the 3' UTRs of C. elegans transcripts and explore their heterogeneity in different developmental stages and tissues.
The general scheme for our RACE experiments is shown in the figure. For our 5' RACE experiments we made use of the trans-spliced SL1 and SL2 leader sequences, instead of ligating a universal sequence to 5' of the transcripts. Approximately 70% of all C. elegans mRNAs have a trans-spliced leader sequence. The great majority of these correspond to a 22 base-long sequence known as "SL1," with "SL2" being the next most frequent, making up 15% of the worm's trans-spliced leaders. The use of SL1/SL2, as opposed to the ligation of an arbitrary sequence to the transcripts' 5' ends has the following advantages: i) no additional manipulation of RNA is needed, ii) the presence of SL1 on a mRNA ensures that the mRNA has an intact 5' end and is full length.nnTo generate the RACE fragments, we reverse transcribed total C. elegans RNA (isolated from mix-stage, asynchronously growing N2 worm population) using either dT16 (for 5' RACE) or used our tailed dT primer (for 3' RACE). Nested PCRs were performed to increase sensitivity and specificity. The generated PCR products were then cloned recombinationally and sequenced from the 5' end, generating "RACE Sequence Tags" (or "RSTs").nnThe RSTs included in this searchable database are vector and quality trimmed (SL and poly(A) sequences were not removed from RSTs). In quality trimming, the first sliding window of 20 nt long with an average quality score higher than 15 marks the start of good quality sequences. Likewise, the first sliding window of 20 nt with average quality score lower than 15 marks the end of good quality sequences.