Dissertation/Thesis Abstract

The embryonic mir-35 microRNA cluster regulates development and RNAi efficiency in Caenorhabditis elegans
by Massirer, Katlin B., Ph.D., University of California, San Diego, 2009, 114; 3378694
Abstract (Summary)

MicroRNAs (miRNAs) are ∼22 nucleotide small RNAs that regulate gene expression by pairing with partial complementarity to target mRNAs. While loss of specific miRNAs can result in distinct phenotypic abnormalities, very few miRNA genes have been shown to be essential for viability. In this dissertation, I demonstrate that the mir-35 gene in Caenorabditis elegans has a vital role in embryogenesis. This gene encodes a cluster of 7 paralogous miRNAs, mir-35-41, that are highly expressed in C. elegans embryos. I show that loss of the mir-35-41 cluster in the genetic mutant mir-35(gk262), results in embryonic lethality, with defects in cytokinesis during the first embryonic cellular divisions and delays in cell cycle progression during subsequent embryonic cell divisions. Consistent with a role for mir-35 miRNAs in early embryogenesis, the inviability of mir-35(gk262) worms was rescued maternally, suggesting that deposition of the miRNA gene products is sufficient for embryogenesis. Supporting this idea, the precursor and mature forms of mir-35 are detectable in female worms that produce only oocytes. Additionally, accumulation of mature mir-35 miRNA correlated with the production of embryos. Taken together, my results suggest that RNA products expressed by the mir-35 gene are deposited in oocytes; following fertilization, increased maturation and synthesis of mir-35-41 miRNAs are then available to control early embryonic events, including cytokinesis and cell cycle progression.

In the second part of my dissertation, I investigated a surprising link between the mir-35 gene and the RNAi pathway. While performing RNA interference (RNAi) experiments in mir-35(gk262) worms, I observed strong RNAi hypersensitivity of the strain. The hypersensitivity is dependent on the canonical RNAi pathway and is similar in levels to the described lin-35 mutant. Additionally, microarrays indicated overlap in gene regulatory pathways for mir-35 and lin-35. I found that LIN-35 protein levels are significantly reduced in mir-35(gk262) embryos, indicating that the mir-35-41 miRNAs positively regulate accumulation of LIN-35 protein. Although the regulation is probably indirect, the decreased level of LIN-35 likely explains the RNAi hypersensitive phenotype of mir-35 mutant worms. Importantly, lin-35 encodes the worm homolog of the human Rb retinoblastoma gene. Another connection between the mir-35 gene and the RNAi pathway is the unexpected finding that nonspecific dsRNA affects the viability of mir-35(gk262) mutants. In contrast to almost complete lethality observed in the absence of mir-35-41 miRNAs at the restrictive temperature, I found that the introduction of non-specific dsRNA could partially rescue this lethality. Since the rescue is dependent on RNAi pathway genes, my results indicate that mir-35-41 is required for embryonic viability in a pathway that can be compensated by the initiation of RNAi.

In conclusion, my research demonstrates that the mir-35-41 miRNAs are important for embryonic viability and regulate the efficiency of RNAi in C. elegans. Although the RNAi hypersensitivity of mir-35 mutants may be largely through down-regulation of LIN-35 in these mutants, the mir-35-41 miRNAs regulate lin-35 and other genes in parallel pathways important for embryogenesis. In support of this model, embryos with loss of lin-35 alone are viable, with loss of mir-35 alone have reduced viability and with loss of both genes are inviable. My work establishes a new regulator of lin-35 and demonstrates novel connections between RNAi pathway genes and embryonic viability.

Indexing (document details)
Advisor: Pasquinelli, Amy E.
Commitee: Bafna, Vineet, Kolodner, Richard, Niwa, Maho, Oegema, Karen, Rosenfeld, Michael G.
School: University of California, San Diego
Department: Biology w/Spec Bioinformatics
School Location: United States -- California
Source: DAI-B 70/11, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Genetics, Evolution and Development
Keywords: Embryonic development, Lin-35, RNAi, microRNA, mir-35
Publication Number: 3378694
ISBN: 9781109425925
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