The C. elegans pocket protein ortholog, LIN-35, plays an important role in a number of pathways, including those responsible for cell cycle control, developmental fate, and morphogenesis. Using a previously described screen for
lin-35-synthetic mutants, we isolated a mutation in Y59A8B.13 (referred to as
slr-2). Sequencing of this mutation shows that it causes a transition in the 3 splice site of the fourth intron, resulting in both truncation of the protein and a dramatic decrease in mRNA levels. Homozygous
lin-35;
slr-2 double mutant animals have a synthetic early larval arrest phenotype and show evidence of starvation, despite having no gross morphological defects preventing ingestion. The capability of these animals to internalize fluorescent beads was assessed, and further argues that
lin-35;
slr-2 mutants are likely to be physically able to consume bacteria, although they may be deficient in nutrient absorption downstream of mechanical disruption. This conclusion is also supported by the ability of axenic medium to provide partial rescue of the
lin-35;
slr-2 larval arrest phenotype.
slr-2 encodes a zinc finger protein with a C-terminal trans activation domain, suggesting that it may play a role in gene regulation. Transcriptional fusion of a GFP reporter with the
slr-2 promoter demonstrated that the gene was nearly ubiquitously expressed in embryos, but has a more limited expression profile in larvae and adults, where mostly intestinal and some neural expression was seen. Although this data provided an idea of where the dysfunction may occur, it did not clearly identify the defect. Therefore, a microarray analysis of
slr-2 single mutant worms was performed. The majority of responsive genes were downregulated, including critical genes in a number of metabolic pathways such as the TOR and insulin-receptor pathways, suggesting that
slr-2 worms may be deficient in nutrient conversion. Genes located in intestinal and neurological mountains (groups of coregulated genes described by Kim, et al., 2001) were upregulated, arguing that
slr-2 normally represses these genes. This is also consistent with GFP reporter assays. Also, a highly significant overlap with a SAGE gut library was observed, suggesting that
slr-2 plays a role in the expression of genes within the intestine of these animals, which is correlated with its expression. Some overlap between differentially expressed genes in
slr-2 and
lin-35 mutants was also noted, primarily in genes involved in intestinal and neurological roles, although the specific genes causing the synthetic phenotype have yet to be identified. Together, these data suggest that the observed larval arrest is at least partially due to a defect in nutrient consumption.