MicroRNAs (miRNAs)

The recent discovery of miRNAs revolutionized our understanding of gene control. Genetic studies in the nematode Caenorhabditis elegans (Figure 1) revealed the first members of what we now recognize as an extensive family of regulatory RNAs that exist in all multicellular organisms (Pasquinelli & Ruvkun, 2002). 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 cancer and other diseases (Pasquinelli et al., 2005). 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 potential 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) (Reinhart et al., 2000). Prior to the revelation that let-7 encoded a tiny RNA, only one other such gene, called lin-4, had been known in any organism, and it too regulated development in C. elegans (lin = lineage, which refers to its role in regulating cell lineage patterns of division) (Lee et al., 2003). The let-7 RNA turned out not to be restricted to worms, and instead was found to be expressed in many different animal species, including humans (Pasquinelli et al., 2000). Soon after the realization that 22 nucleotide RNA genes lurk beyond the worm genome, hundreds of other such genes were discovered, and they now constitute the miRNA class of regulatory RNAs (Pasquinelli, 2002). Based on the genetically defined functions of the founding miRNA genes, lin-4 and let-7, miRNAs generally inhibit expression of specific protein-coding genes by base-pairing to the messenger RNAs (mRNAs). For example, the let-7 miRNA regulates expression of the lin-41 gene by recognizing two let-7 complementary sites (LCS’s) in the 3’ untranslated region (UTR) of the lin-41 mRNA (Reinhart et al., 2000; Slack et al., 2000). The mechanism used by miRNAs to recognize and regulate specific target genes is yet to be unraveled.

My lab is particularly interested in the let-7 miRNA for two primary reasons. First, we hope that by understanding how expression of this miRNA is regulated and how it controls its targets, we may learn general rules that help elucidate miRNA function. Second, we aim to uncover the biological pathways regulated by this strikingly conserved miRNA gene. In C. elegans, the let-7 gene regulates development and cellular differentiation and this function is likely to be related to its recently proposed role as a tumor suppressor in humans (for example see Johnson et al., 2005).

Questions that direct our research:

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 (Figure 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. In a collaborative effort by several lab members, we identified the complete let-7 pri-miRNAs and learned that transcription of this miRNA gene initiates hundreds of nucleotides upstream of the mature miRNA sequence (Bracht et al., 2004). This finding now positions us to uncover the transcriptional regulatory elements that determine when and where this miRNA will be expressed. Surprisingly, we found that the let-7 pri-miRNA transcripts undergo splicing and this step appears to be important for downstream processing events (Bracht et al., 2004). A nuclear complex containing the RNAse Drosha and its RNA binding partners is responsible for clipping the stem-loop precursor (pre-miRNA) from the longer pri-miRNAs, but how this complex recognizes its substrate is yet to be fully understood (Figure 2). Two PhD graduate students in the lab, John Bracht and Katlin Massirer, are studying additional miRNA genes with the goal of identifying general features important for pri-miRNA transcription and processing. John has also developed a genetic screen to uncover elements in the let-7 gene as well as protein factors that regulate processing of let-7 RNA and perhaps miRNAs in general.

How do miRNAs regulate gene expression? In most cases, animal miRNAs regulate specific genes by partially base-pairing to complementary sequences in the messenger RNAs (mRNAs) of protein-coding genes (Figure 2). The human genome contains over 300 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 focus on specific miRNA genes in C. elegans and use molecular and genetic experiments to identify potential targets. We also subject these candidate target genes to bioinformatic analyses to uncover regulatory motifs. In most cases, miRNAs cause down-regulated expression of their target genes, but the mechanism of this inhibition is not well understood. Through a group effort, the Pasquinelli lab showed that regulation by miRNAs can result in degradation of the target mRNA (Figure 2) (Bagga et al., 2005). This was the first demonstration that endogenous gene regulation by miRNAs that partially base pair to their targets involved destabilization of the mRNAs.

Presently, the role of translational repression and mRNA degradation in regulating specific miRNA targets is unclear. Dr. Shveta Bagga, a post doctoral associate in the lab, aims to understand the mechanism of miRNA action by focusing on the let-7 miRNA and its known targets in C. elegans. She uses a battery of molecular and biochemical approaches to identify proteins and features of the miRNA and target important for mediating the regulation. Brad Hehli, a PhD graduate student in the lab, and Ken Finn, an undergraduate Biology Honors Student, are studying RNA binding proteins and other factors that are miRNA effector candidates. By studying defined miRNA and target pathways in C. elegans, the Pasquinelli lab hopes to unravel the novel modes of gene regulation guided by miRNAs.

What is the biological function of miRNA regulatory pathways? Establishment of the precise gene regulatory role of miRNAs in any biological pathway is complicated by the fact that animal miRNAs typically recognize mRNA target sites that lack perfect complementarity. Thus, the basic problem of how miRNAs use limited sequence complementarity to regulate specific mRNAs is an outstanding challenge in the field. We are performing molecular and genetic experiments to identify direct targets of specific miRNAs. Shaun Hunter, Katlin Massirer and Brad Hehli, all PhD graduate students in the lab, are carrying out differential RNA analyses to identify genes mis-regulated when specific miRNA pathways are defective. For example, Shaun has uncovered genes up-regulated in let-7 mutant worms that are potentially direct targets of regulation by this miRNA. We use computational methods to predict sequences that mediate targeting of the genes regulated by the miRNA pathway. Expanding the list of bona fide miRNA targets will give insights into miRNA/ target interactions and lead to improved computational predictions. By focusing on a select set of genes that we can experimentally validate as direct targets of miRNA control, we hope to uncover general rules for how miRNAs recognize and regulate their target genes.

Some miRNA genes, like let-7, are essential for normal development (Figure 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. A goal of our studies on the worm let-7 gene is to understand the broad role let-7 plays in cellular differentiation events across species. Shaun Hunter, Zoya Kai, PhD graduate students in the lab, and Janette Holtz, our lab manager, are working to elucidate the gene regulatory networks controlled by let-7 miRNA. Using genetic and molecular experiments, they aim to understand the role of let-7 targets and why these genes need to undergo regulation by let-7 and the miRNA pathway at precise times in development.

Outlook

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. 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. Recent 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 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.

Referenced Literature

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(4):553-63.

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.

Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ. RAS is regulated by the let-7 microRNA family. Cell. 2005 120(5):635-47.

Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993 75(5):843-54.

Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000 408(6808):86-9.

Pasquinelli AE. MicroRNAs: Deviants No Longer. Trends in Genetics. 2002 April; 18(4):171-173.

Pasquinelli AE, Ruvkun G. The Role of Heterochronic Genes in Development and Evolution. Annu Rev Cell and Dev Biol. 2002 18:495-513.

Pasquinelli AE, Hunter S, Bracht J. MicroRNAs: a developing story. Curr Opin Genet Dev. 2005 15(2):200-5.

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.

Slack FJ, Basson M, Liu Z, Ambros V, Horvitz HR, Ruvkun G. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol Cell. 2000 5(4):659-69.