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

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.