MicroRNAs (miRNAs)


The recent 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 (Pasquinelli & Ruvkun 2000, Pasquinelli 2002 & 2005 & 2012, Aalto & Pasquinelli 2012; Mondol & Pasquinelli 2012).  Already there is evidence that specific miRNAs 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 (Pasquinelli et al., 2005, Massirer & Pasquinelli 2006, Godshalk et al., 2010; Mondol & Pasquinelli 2012). 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 miRNA


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) (Reinhart et al., 2000). The ~22 nucleotide let-7 RNA is expressed in many different animal species, including humans (Pasquinelli et al., 2000), 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 (Boyerinas et al., 2010).  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 2).  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, including a novel autoregulatory loop where mature let-7 miRNA regulates its own processing (Bracht, Hunter, et al., 2004, Van Wynsberghe et al., 2011, 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 2).  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.  The human genome contains over 700 different miRNA genes, each of which may directly regulate hundreds of protein coding genes.  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).  This work revealed defined Argonaute binding sites on a global scale and the challenge now is to match specific miRNAs with the targeted sequences.  Additionally, we discovered unexpected Argonaute binding sites that have revealed new functions of the miRNA pathway (Zisoulis, Kai et al., 2012).





What is the biological function of miRNA regulatory pathways?


Some miRNA genes, like let-7, are essential for normal development (FIG 1).  The let-7 miRNA and its temporally regulated expression pattern are widely conserved across animal phylogeny and misexpression of this miRNA has been linked to cancer in humans.  Through genome wide expression studies and genetic analyses, we have identified new genes in the let-7 pathway that expand the network controlled by this essential miRNA (Hunter et al., in prep). A goal of our studies on the worm let-7 gene is to understand the broad role this miRNA plays in cellular differentiation events across species.


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.





Outlook


The discovery of miRNA genes in C. elegans and the subsequent recognition that this family of RNAs extends throughout most 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. There are diverse types of non-coding RNAs that control stress responses in bacteria, chromosome segregation in yeast, flowering in plants, and viral replication in animal cells.  The indication that well over half of the human genome is transcribed raises the possibility that non-coding RNA genes may begin to rival 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 to 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.




References


Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, Eachus R, Pasquinelli AE. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation.  Cell. 2005 122:553-563.


Boyerinas, B., Park, S.-M., Hau, A., Murmann, A.E., Peter, M.E., 2010. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer 17, F19-36.


Bracht J, Hunter S, Eachus R, Weeks P, Pasquinelli AE.  Trans-splicing and polyadenylation of let-7 microRNA primary transcripts. RNA. 2004 10:1586-1594.


Bracht JR, Van Wynsberghe PM, Mondol V, Pasquinelli AE. Regulation of lin-4 miRNA Expression, Organismal Growth and Development by a Conserved RNA Binding Protein in C. elegans. Dev Biol. 2010 Dec 15;348(2):201-21.


Chendrimada TP, Finn KJ, Ji X, Baillant D, Gregory RI, Liebhaber SA, Pasquinelli AE, Shiekhattar R. MicroRNA silencing through RISC recruitment of eIF6.  Nature 2007 447:823-828.


Godshalk SE, Melnik-Martinez KV, Pasquinelli AE, Slack FJ. MicroRNAs and cancer: a meeting summary of the eponymous Keystone Conference. Epigenetics. 2010 Feb;5(2):164-8.


Holtz J, Pasquinelli AE. Uncoupling of lin-14 mRNA and protein repression by nutrient deprivation in Caenorhabditis elegans. RNA. 2009 15:400-5.


Hunter S, Finnegan E, Kai ZS, Zisoulis, DG, Melnik-Martinez KV, Pasquinelli AE. A network of let-7 targets regulates cellular differentiation in C. elegans. In preparation


Massirer K, Pasquinelli AE. The evolving role of microRNAs in animal gene expression. BioEssays. 2006 28:449-452.


Massirer KB, Perez SG, Mondol V, Pasquinelli AE. The miR-35-41 family of microRNAs regulates RNAi sensitivity in Caenorhabditis elegans. PLoS Genetics. 2012 Mar;8(3):e1002536.


Mondol V, Pasquinelli AE. Let’s make it happen:  The role of let-7 microRNA in development. In Eran Hornstein, editors: MicroRNAs in Development, CTDB, UK: Academic Press, 2012, 99:1-30.


Pasquinelli AE, Ruvkun G. Control of Developmental Timing by MicroRNAs and Their Targets. Annu Rev Cell Dev Biol. 2002 18:495-513.


Pasquinelli AE. MicroRNAs:  Deviants No Longer. Trends Genet.  2002 18:171-173.


Pasquinelli AE. MicroRNAs:  a Small Contribution from Worms.  In RNA Interference Technology: From Basic Science to Drug Development, ed. K Appasani. Cambridge, UK: Cambridge University Press. 2005. p69-83.


Pasquinelli AE, Hunter SE, Bracht J. MicroRNAs:  A Developing Story. Curr Opin Genet Dev. 2005 15:200-205.


Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000 403:901-906.


Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE. Co-transcriptional association of LIN-28 with let-7 primary transcripts regulates miRNA maturation in C. elegans. Nat Struct Mol Biol. 2011 Mar;18(3):302-8.


Zisoulis DG, Lovci MT, Wilbert ML, Hutt KR, Liang TY, Pasquinelli AE, Yeo GW. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat Struct Mol Biol. 2010 17:173-9.


Zisoulis DG, Yeo GW, Pasquinelli AE. Comprehensive identification of miRNA target sites in live animals. Methods Mol Biol. 2011 732:169-85.


Zisoulis DG, Kai ZS, Chang RC, Pasquinelli AE. Auto-regulation of miRNA biogenesis by let-7 and Argonaute. Nature 2012 Jun 28;486(7404):541-4.