DEVELOPMENTAL TIMING AND TRANSLATIONAL REGULATION IN THE NEMATODE CAENORHABDITIS ELEGANS

ERIC G. MOSS, Ph.D., Associate Member

This laboratory examines the unusual genetic regulatory mechanisms that control gene expression and cell behavior in the simple animal, C. elegans. We are primarily interested in mechanisms that operate at the level of mRNA translation because the mechanistic basis of this kind of regulation is largely unexplored. We believe that understanding mRNA translation control in this model system will provide insight into how it proceeds in human cells, and potentially yield new ways of intervening in gene expression.

The main focus of our work is the developmental timing system of the nematode. We are investigating how the events of larval development are made to occur in the appropriate order and in synchrony throughout the animal. A number of mutant nematode strains have been isolated that are defective in developmental timing. These mutants have defined a group of genes collectively referred to as the "heterochronic genes." The gene lin-4 encodes a 22-nucleotide RNA that is a repressor of the genes, lin-14 and lin-28, which are positive regulators of each other and act on as yet unidentified targets. A new heterochronic gene, lin-46, may function downstream of these genes, or may be involved in the mutual regulation of lin-14 and lin-28.

We aim to identify the molecular relationships among the products of these genes and define how they act at the biochemical level. In one instance, where our work may have a direct connection to human cancer, we are attempting to define the role of a small family of RNA-binding proteins in the control of multidrug resistance, an important problem in chemotherapy.

For developmental timing to occur normally in C. elegans, the product of the lin-28 gene must be expressed in the first larval stage and shut off by the second stage. If lin-28 remains inappropriately expressed at later times, due to a mutation in its regulatory mechanism, then a retarded developmental phenotype results. Retarded development is where events of an earlier stage are reiterated at the expense of late stage events. If lin-28 is insufficiently active, then a precocious phenotype results where stage-specific events are skipped and otherwise later events occur early.

Our genetic analysis has determined that two regulatory circuits act on the expression of lin-28 to ensure its proper regulation: one is a negative regulation by lin-4 and another is a positive regulation by lin-14. The lin-4 RNA acts directly on the mRNA of lin-28, requiring a 15-nucleotide segment in the 3 untranslated region (UTR) of lin-28 mRNA. We are attempting to determine whether there are additional regions in the 3 UTR that are necessary for this regulation, or whether this 15-nucleotide element is sufficient. The direct effector of the lin-14 positive regulatory circuit is not known, however, the regulation appears also to occur through the 3 UTR of the lin-28 mRNA. We are mapping the region responsible for this regulation as a prelude to identifying the factor that mediates the regulation.

The regulation of lin-28 by lin-4 is a naturally occurring example of "antisense" regulation (Figure 1). Understanding the mechanism of this regulation may help us design novel antisense strategies. We have found that lin-4 affects the translation of the lin-28 mRNA, but does not appear to act by causing degradation of the mRNA. In fact, we have found that the lin-28 mRNA remains associated with the translation machinery after lin-4 has repressed its expression. We are exploring the possibility that lin-4 is responsible for directing base-specific modification of the lin-28 mRNA, and are seeking protein factors that lin-4 may recruit to the site of duplex formation between the two RNAs.

THE ROLE OF THE lin-46 GENE IN DEVELOPMENT. MOSS

As a way of finding new heterochronic genes, we have sought mutations that suppress the defects caused by mutations in the genes lin-14 and lin-28. As a result, we have identified a new gene, lin-46. Mutations in this gene are able to suppress the precocious phenotypes of all mutant alleles of lin-28 and certain alleles of lin-14, but curiously, not lin14 null alleles. On their own, lin-46 mutations display a weak retarded phenotype revealed by reiterated cell divisions in hypodermal cells. lin-46 is clearly important in the developmental timing mechanisms, however, its precise role is very difficult to deduce from the genetic analysis performed thus far. We have cloned the DNA encoding lin-46 and determined that the gene product belongs to a family of proteins, found from bacteria to humans, that have diverse roles in metabolism and cell behavior. Based on analogies to the functions of these proteins, we are pursuing a biochemical function for the lin46 gene product.

REGULATION OF MULTIDRUG RESISTANCE. PORAT

Work by others has indicated that multidrug resistance in human cancer cells correlates with the increased expression and nuclear localization of a class of RNA-binding proteins, Y-box factors. These factors share a nucleic acid binding motif (a CSD domain) with the product of the heterochronic gene, lin-28. As part of our general interest in this class of RNA-binding proteins, we are seeking to understand whether, and how, the Y-box factors regulate the expression of the P-glycoproteins, which mediate multidrug resistance.

We have found that C. elegans is sensitive to a number of chemotherapeutic drugs that are substrates for P-glycoproteins, including daunorubicin and vincristine. We have developed a quantitative bioassay based on the three-day development of the nematode to measure the sensitivity of the animal to the drugs' effects. Furthermore, we have found that a mutant strain, which lacks three Pglycoprotein genes, is significantly more sensitive to certain drugs than the wild type strain (Figure 2). This finding demonstrates that, as in humans, these proteins are responsible for the animal's resistance to these drugs. Our plan is to manipulate the expression of the Y-box factors using transgenic and knockout technologies to determine whether they can affect expression of P-glycoproteins and drug resistance.

The complexity of the heterochronic regulatory pathway and the diversity of the molecules encoded by these genes suggest that there are more such factors remaining to be discovered. To identify new heterochronic genes, we are using the approach of screening for genetic suppressors and enhancers of existing heterochronic mutations. So far, we have found one new allele and are in the process of determining whether it is an allele of a previously identified heterochronic gene.

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998