Conserved elements associated with ribosomal genes and their trans-splice acceptor sites in Caenorhabditis elegans

Abstract

The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression.

Figure 1.

Ribosomal instances of the motif group 12-0. The motif group 12-0 was found upstream of 28 ribosomal transcripts, of which two pairs were on bidirectional promoters: lsm-1 (F40F8.9; a small nuclear ribonucleoprotein splicing factor) and rps-9 (F40F8.10) and rps-30 (C26F1.4) and rpl-39 (C26F1.9). Shown here are the 26 ribosomal upstream regions; instances of motif group 12-0 are shown in red. Instances of motif groups 12-1, 12-5, 12-8, 12-11 and 12-18 are shown in cyan, magenta, gray, blue and green, respectively. The motif logo for all instances of motif group 12-0 in these regions is also shown.

Figure 2.

Schematic of GFP constructs. For each gene with both previous GFP constructs and an instance of motif group 12-0 in its upstream region, three constructs were made. The first construct consisted of the gene’s upstream region up to and including the motif but no further (primers indicated by yellow and purple arrows), the second construct was slightly shorter such that the motif was excluded (primers indicated by cyan and purple arrows) and, for the third construct, we introduced a mutation in the central CG of the motif via a primer (primers indicated by orange and purple arrows). Results of the GFP expression assays are shown in Table 6.