The discovery of microRNAs (miRNAs) has revolutionized our understanding of gene control. Genetic studies in the nematode Caenorhabditis elegans (FIG 1) revealed the first members of what we now recognize as an extensive family of regulatory RNAs that exist in most multicellular organisms (reviewed in Pasquinelli 2002 & 2012; Massirer & Pasquinelli 2006; Aalto & Pasquinelli 2012). Specific miRNAs have been found to play key roles in controlling development, stem cell fates and neuronal differentiation, and mutations in human miRNA genes have been linked to oncogenic and other disease states (reviewed in Pasquinelli et al., 2005; Godshalk et al., 2010). The Pasquinelli lab couples C. elegans genetics with molecular and biochemical techniques to understand the basic mechanisms of miRNA expression and function and to elucidate the biological roles of specific miRNAs in cellular differentiation programs.
The let-7 microRNA
The let-7 miRNA gene is exceptional in the conservation of its sequence and function. The let-7 miRNA was first discovered in Gary Ruvkun’s lab as a gene essential for development in C. elegans worms (let = lethal, which refers to the premature lethality of worms deficient for this gene) (FIG 1) (reviewed in Mondol & Pasquinelli 2012). The 22 nucleotide let-7 RNA is expressed in many different animal species, including humans (Pasquinelli et al., 2000) (FIG 2), where it regulates key cell division and differentiation pathways. The let-7 miRNA is considered a tumor suppressor, as it has been demonstrated to halt tumor progression in lung and breast cancer models (reviewed in Mondol & Pasquinelli 2012). Study of the let-7 miRNA is of particular interest for two primary reasons. First, by understanding how expression of this miRNA is regulated and how it controls its targets, general insights into miRNA biogenesis and function will be gained. Second, uncovering the biological pathways regulated by this strikingly conserved miRNA gene will help elucidate the role of let-7 in human health and disease.
How is the expression of miRNAs regulated?
MiRNA genes typically encode long primary transcripts (pri-miRNAs) that undergo multiple processing steps to generate the mature ~22 nucleotide miRNA (FIG 3) (reviewed in Finnegan & Pasquinelli 2013). Many miRNA genes are expressed at precise times in development and in specific tissues. To understand how these temporal and spatial expression patterns are achieved, we study the transcriptional and processing events that cooperate to produce specific miRNAs at the right time and in the right place. Since aberrant expression of specific miRNAs has been linked to many different diseases, we have a deep interest in determining the molecular mechanisms that control miRNA biogenesis at every step from transcription, to processing, to stabilization of the mature miRNA. Our studies of let-7 have revealed dynamic transcriptional and post-transcriptional mechanisms that control the accumulation of this miRNA (Bracht, Hunter, et al., 2004; Van Wynsberghe et al., 2011; Kai et al., 2013; Van Wynsberghe et al., 2014), including a novel autoregulatory loop where mature let-7 miRNA regulates its own processing (Zisoulis, Kai et al., 2012). We found that expression of another miRNA, lin-4, is also regulated at multiple levels through mechanisms that are different from those used to control let-7 (Bracht, Van Wynsberghe et al., 2010). Further investigation of these miRNAs, as well as others, will elucidate how miRNA biogenesis is controlled in a developing organism and reveal how this can go wrong in disease states.
How do miRNAs regulate gene expression?
MiRNAs regulate specific genes by partially base-pairing to complementary sequences in the messenger RNAs (mRNAs) of protein-coding genes (FIG 3) (reviewed in Pasquinelli 2012). We have shown that regulation of miRNA targets in C. elegans results in mRNA degradation, requires specific factors to translationally repress and destabilize the mRNA and is sensitive to environmental conditions (Bagga et al., 2005, Chendrimada et al., 2007, Holtz & Pasquinelli 2009). However, the detailed mechanism of how specific targets are recognized and regulated is still under investigation. Since animal miRNAs use partial base-pairing to bind mRNA target sites, identifying biologically relevant targets of specific miRNAs has been a great challenge. To help elucidate how miRNAs find and regulate targets with limited sequence complementarity, we have performed genome wide analyses to identify endogenous targets of miRNA regulation (Zisoulis et al., 2010 & 2011; Hunter, Finnegan et al., 2013; Broughton & Pasquinelli 2013). This work provided the first map of Argonaute binding sites on a global scale in a live organism, and the challenge now is to match specific miRNAs with the targeted sequences. Additionally, we discovered unexpected Argonaute binding sites that may reveal new functions of the miRNA pathway (reviewed in Pasquinelli 2013).
What is the biological function of miRNA regulatory pathways?
Some miRNA genes, like let-7, are essential for normal development (FIG 1). The miR-35-41 family of miRNAs is important for embryogenesis and, unexpectedly, we found that these miRNAs also regulate the effectiveness of RNA interference (RNAi) in worms (Massirer et al., 2012). Although, the majority of other worm miRNAs appear to be dispensable during worm development, we have uncovered key roles for the miRNA pathway during stress and lifespan determination. Current projects in the lab seek to discover the specific miRNAs, cofactors and mechanisms that control the organismal response to environmental perturbations and aging.
We are also interested in the miR-35-41 family of miRNAs, which are essential for embryogenesis. While studying the role of these miRNAs during this stage in development, we made the surprising discovery that miR-35-41 miRNAs regulate RNAi pathways in C. elegans (Massirer et al., 2012). In mir-35-41 mutants, RNAi sensitivity is enhanced and endogenous pathways that utilize small RNAs are defective. This finding demonstrates that miRNAs can broadly regulate other small RNA pathways and, thus, have far-reaching effects on gene expression beyond directly targeting specific mRNAs.
The discovery of miRNA genes in C. elegans and the subsequent recognition that this family of RNAs extends throughout all multicellular organisms has provided researchers with much more than a new class of regulatory RNAs. Although non-coding RNAs have long been appreciated as essential for core biological processes, such as protein translation and mRNA splicing, it is now evident that RNA genes are much more extensive in number and function. The finding that well over half of the human genome is transcribed raises the possibility that non-coding RNA genes may even surpass protein-coding genes in number and perhaps in functional diversity. The recent explosion of interest in RNA-mediated gene regulatory mechanisms is also bolstered by the promise for development of RNA therapeutics to specifically inactivate oncogenes or viruses, for example. This potential depends on basic research aimed at deciphering the elegant regulatory mechanisms evolution has bequeathed to RNA. Thus, a broad goal in the Pasquinelli lab is to contribute experimental evidence towards the general understanding of how regulatory RNAs control gene expression. We hope this knowledge will help elucidate the roles of RNA genes in human health and disease and will provide groundwork for RNA based medical applications.