Since their discovery a little over two decades ago, microRNAs have emerged as pervasive, conserved, post-translational regulators of gene expression. In the Kim lab we study how microRNAs control fundamental biological processes including cellular differentiation, post-embryonic development, and oncogenic cell proliferation. We are interested in addressing the question,
“What regulates microRNA function?”
To identify and characterize factors involved in microRNA regulation the Kim lab employ a combination of genetic, genomic, biochemical, and proteomic techniques. The robust and facile genetics of C. elegans and the ability to use it in high-throughput methods make it an attractive in vivo system for this task. Historically C. elegans has been an important model system for the study of microRNAs: the first miRNAs, lin-4 and let-7, were discovered in genetic screens for factors that controlled the timing of cell division and differentiation during C. elegans development (Lee et al., 1993; Wightman et al., 1993; Reinhart et al., 2000). Work from our lab has identified a host of potential factors that may influence microRNA function: A genome-wide RNAi screen in C. elegans identified many candidate genes that await characterization (Kim et al., 2005). We have also identified novel proteins that interact with microRNA pathway proteins by mass spectrometry. These two complementary approaches recently led to our discovery that the highly conserved serine/threonine kinase Casein Kinase II (CK2) facilitates microRNA silencing by modification of the microRNA protein co-factor.
Two areas of active microRNA research in the Kim lab are (1) elucidating the role of post-translational modifications to proteins that function in microRNA biogenesis and silencing, and (2) investigating how RNA-binding proteins affect silencing by the ribonucleoprotein microRNA effector complex. We are also interested in deciphering how microRNA regulation is modulated under adverse conditions, including environmental stress. Since the microRNA pathway is highly conserved from human to worm our findings have the potential to translate to the eventual treatment of human disease as microRNA dysregualtion is a hallmark of cancer and is implicated in a variety of other human pathologies.
II. Endogenous small siRNAs
Endogenous small RNA pathways in C. elegans, including siRNAs and piRNAs, are required for critical germline processes including repression of invasive genomic elements, maintaining an epigenetic memory of gene expression, and orchestrating heterochromatin deposition at target loci. The significance of these functions is highlighted by the severe fertility phenotypes observed when these small RNA classes are lost, as loss of piRNAs leads to temperature-sensitive sterility and loss of nuclear siRNAs leads to progressive sterility (germline mortality).
In order to understand how these pathways are regulated and why they are so critical for germline function, we are using genetic, molecular, and biochemical techniques to identify novel components of these small RNA pathways and to establish a mechanistic connection between small RNAs and chromatin organization and genome defense. Current projects include (1) Identification of factors controlling differential expression of small RNA classes in male versus female germline, (2) characterizing the role of endogenous siRNAs in transgenerational chromatin organization, and (3) identification of novel factors promote or suppress endogenous siRNA effector functions.
III. RNA Binding Proteins
RNA-binding proteins (RBPs) are crucial components of a diverse range of cellular processes. They serve critical roles in post-transcriptional gene regulation (PTGR) by assembling RNAs and other protein co-factors into higher-order ribonucleoprotein (RNP) complexes to regulate RNA splicing, transport, storage, stability, and translation. Our lab has recently begun to characterize diverse PTGR roles for the conserved Puf (Pumilio/FBF) family of RBPs in yeast and nematodes in the context of how they contribute to regulation of environmental stress responses, germline stem cell maintenance, and embryonic development. In addition, we have identified hundreds of novel RBPs in these model organisms and are actively seeking explanations for why these proteins – with primary roles in transcription, cellular trafficking, and metabolism, among others – might posses RNA-binding abilities. To understand the specific roles of novel RBPs, our lab combines classical biochemical and molecular genetics approaches with high-throughput crosslinking and deep sequencing of direct RNA target sites to discover how RBP:RNA interactions affect RNA translation, stability, localization, and degradation.