ROLE OF DICER IN miRNA/RNAi PATHWAYS AND REGULATION OF miRNA REPRESSION Witold Filipowicz Friedrich Miescher Institute for Biomedical Research, P.O. Box 2543, 4002-Basel, Switzerland
[email protected] INTRODUCTION. MiRNAs are ~20-nt-long regulatory RNAs operating in matazoan animals and plants. In animals, expression of many miRNAs is tissue or development specific, and major changes in miRNA expression are observed in human pathologies. MiRNAs regulate gene activity post-transcriptionally, by imperfectly base-pairing to the 3’-UTR of mRNAs, what results in translational repression or mRNA degradation. MiRNA biogenesis and function require a set of dedicated proteins. Dicer and Argonaute (Ago) proteins are the best-characterized components of the miRNA machinery, also active in the RNA interference (RNAi) pathway. Human Dicer functions in a complex with a double-stranded RNA (dsRNA) binding protein TRBP, identified previously as a regulator of protein kinase PKR and a protein intreacting with the HIV-1 TAR. Dicer cleaves pre-miRNAs by recognizing the base of the pre-miRNA hairpin and measuring the ~20-bp distance from the hairpin end. It cuts off siRNAs from the end of dsRNA substrates by a similar distance-measuring mechanism (1). RESULTS AND DISCUSSION. Mammalian genomes encode a single Dicer protein. We will discuss our progress on understanding the function of human Dicer. Dicer is a multidomain endoribonuclease, containing in addition to the RNaseIII-like catalytic domains, a helicase/ATPase, DUF283, PAZ and a C-terminal dsRNA-binding domains. Despite that human Dicer contains a perfect RNA helicase/ATPase domain, in contrast to Drosophila and C. elegans enzymes, it processes dsRNA and miRNA precursors in an ATP-independent way (1). We are investigating contribution of different domains to dsRNA and pre-miRNA binding and to activity of the enzyme in cleaving its substrates. We are also investigating cellular localization of the enzyme and its potential function in the nucleus. Protein partners of human Dicer are being identified by immunoprecipitation and mass spectrometry. We study the mechanism of the miRNA repression in HeLa and hepatoma cells, using mRNAs whose translation is inhibited by endogenous let-7 (ubiquitously expressed) or miR-122 (liver-specific) miRNAs. Studies with reporters, and also endogenous mRNAs such as the mRNA encoding the cationic amino acid transporter (CAT-1), indicated that miRNAs let-7 and miR-122 repress translation at the initiation step. Repressed mRNAs are re-localized to P-bodies, cellular structures involved in storage and degradation of mRNAs. Our results support a two-step mechanism of miRNA action, with the localization to P-bodies being a secondary event, following inhibition of translation (2, 3).
We found that under specific cellular conditions the miRNA-mediated repression can be reversed (3). In response to cellular stress (amino acid deprivation, endoplasmic reticulum or oxidative stress), the repressed CAT-1 mRNA or reporters bearing its 3’UTR exit P-bodies to reassociate with polysomes and to reenter translation (in collaboration with Ellen Closs, Johannes Gutenberg University, Mainz, Germany). Hence, in addition to their established role in mRNA degradation, P-bodies function in storage of the miRNA-repressed mRNAs. The derepression requires binding of HuR, an AU-rich-element-binding protein of ELAV family, to the 3’UTR of target mRNA. We will discuss a possible mechanism of the HuR-mediated derepression. We will also discuss results of collaborative studies on characterization of the in vitro translation system, prepared from mouse Krebs-2 ascites cells (with Nahum Sonenberg et al., McGill University, Montreal). This system recapitulates many features of translational regulation mediated by miRNAs (4). The in vitro data support the notion that let-7 miRNA represses translation at the initiation step. We are also investigating a role of miRNAs during mouse embryonic stem (ES) cell differentiation (collaboration with Mihaela Zavolan, University of Basel). Loss of miRNA pathway components such as Dicer negatively affects differentiation of ES cells, but the underlying molecular mechanisms remain poorly defined. Transcriptome analysis of Dicer-/- cells (kindly provided by Greg Hannon, CSHL) indicated that the ES-specific miR-290 cluster has an important regulatory function in undifferentiated ES cells. Consistently, many of the loss of Dicer defects can be reversed by transfecting miR-290 family miRNAs. We found that Oct-4 silencing in differentiating Dicer-/- ES cells is accompanied by accumulation of repressive histone marks but not by DNA methylation, which prevents the stable repression of Oct-4. The methylation defect correlates with down-regulation of de novo DNA methyltransferases and can be rescued by their ectopic expression or by transfection of the miR-290 cluster miRNAs, indicating that de novo DNA methylation in ES is controlled by miRNAs. REFERENCES 1. Zhang, H., Kolb, FA., Jaskiewicz, L., Westhof, E. Filipowicz, W. (2004) Cell 118, 57-68 2. Pillai, R., Bhattacharyya, S., Artus, C., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E., and Filipowicz, W. (2005) Science 309, 1573-1576 3. Bhattacharyya S, Habermacher, R, Martine, U, Closs, E.I. and Filipowicz, W. Cell 125, 1111–1124 4. Mathonnet, G, Fabian, M.R., Svitkin, Y.V., Parsyan, A., Huck, L., Murata, T., Biffo, S., Merrick, W.C., Darzynkiewicz, E., Pillai, R.S., Filipowicz, W., Duchaine, T.F. and Sonenberg, N. (2007) Science 317, 1764-1767
