Molecular and developmental characterization of functions of the let-7 miRNA and its target LIN-41 in controlling temporal patterning in C. elegans

Abstract

Multicellular organisms start their life as a single cell. During the development of an organism, this single cell proliferates and differentiates forming specialized cells such as neurons, muscle cells, epithelial cells and germ cells. Amazingly, each of these unique cells although having specialized functions, contains an identical genome that is regulated selectively to express a particular set of genes that are specifically required to create a certain cell type. This selective gene expression is the basic principle through which complex multicellular organisms such as humans are formed by repeating two fundamental processes, cell proliferation and cell specialization or differentiation. The regulation of gene expression to increase or decrease the production of specific gene products (protein or RNA) can be modulated at various levels, from transcriptional initiation, to post-transcriptional RNA regulation, and to the post-translational modification of a protein. Misregulation at any of these levels perturb the gene expression, thus resulting in disease state This thesis delves into elucidating how the lethal-7 (let-7) miRNA and its target lineage defective 41 (lin-41) function as post-transcriptional regulators of gene expression. Specifically, I investigated how these genes temporally control gene expression to specify the timing of developmental events in well studied developmental pathway also known as the heterochronic pathway in Caenorhabditis elegans worm. In the first part of this thesis I attempted to uncover a direct interface between the let-7 miRNA and the cell cycle machinery. The goal of this project was to assess how let-7 regulates cell cycle exit and terminal differentiation by regulating target genes directly by binding to their respective 3’UTRs. We performed an RNAi screen against 40 core cell-cycle regulating genes for let- 7 lethality suppression and demonstrated that RNAi against two genes, cdk-1 and cdc-25.2, not only suppressed the let-7 lethality, but also reversed the retarded seam-cell phenotypes, i.e. additional seam cell divisions and alae defects, in let-7(n2853ts) as well as let-7(mn112) animals, confirming specificity of the genetic interaction. However, detailed analysis revealed that although the 3'UTRs of these two mitotic genes might confer posttranscriptional repression, this seems unlikely to be a consequence of let-7 function. Furthermore, we also examined cdk-1::gfp expression and found that knock-down of lin-29, which is downstream of let-7, resulted in elevated levels similar to the effect of let-7 knock-down. Similarly, up-regulation was also observed for RNAi of mab-10, a transcription co-factor that acts in concert with LIN-29 to promote differentiation of the hypodermis. Thus, we conclude that let-7 preferentially regulates cdk-1 indirectly, in a manner that requires the LIN-29 transcription factor. For the second part of this thesis, I focused on lin-41, which is a direct target of let-7. The project involved functional characterization of LIN-41. Specifically in this project we attempted to address two important questions namely how does LIN-41 protein mechanistically regulate gene expression posttranscriptionally and what are its targets. Towards that goal we created a worm strain expressing a functional tagged version of LIN-41. Using the tagged LIN-41, we established its mRNA binding potential by performing LIN-41 coimmunoprecipitation and assessing the mRNA targets of LIN-41 getting pulled down. Our analysis revealed multiple mRNAs i.e. lin-29, mab-10, dmd-3 and mab-3 that associated with the LIN-41 protein. For lin-29 mRNA, we also tested where on the mRNA would LIN-41 protein bind. Our analysis revealed that LIN-41 binds in the 5’UTR region in the lin-29 mRNA and not the 3’UTR region. Additionally our analysis also revealed that the regulation of lin-29 happens at the translational level, by LIN-41 protein binding to lin-29 mRNA in the 5’UTR region. By analyzing worm strains with mutations in the individual domains of LIN-41 we obtained further insight into how LIN-41 may carry out its role as a posttranscriptional regulator of gene expression especially in the context of the somatic tissue development. Thus, we found the NHL domain to be critical for the somatic function of LIN-41. Finally, we tried to identify protein-binding partners of LIN-41 that could play an important role in LIN-41-mediated posttranscriptional gene regulation. However, it remains to be established if the putative protein partners that we found in our analysis are true interaction partners of LIN-41, and if so, how they participate in LIN-41’s functions. Taken together, this work has provided further insight into the functioning of two post-transcriptional regulators of gene expression, let-7 miRNA and its downstream target lin-41.