Portal de Periódicos da CAPES

MicroRNAs: All Gone and Then What?

2005; Elsevier BV; Volume: 15; Issue: 10 Linguagem: Inglês

10.1016/j.cub.2005.05.006

1879-0445

Oliver Hobert,

RNA Research and Splicing

MicroRNAs are abundant gene regulatory factors whose function in animal development and homeostasis is poorly understood. A new study reports the genetic elimination of miRNA function on a full genomic scale and identifies a subfamily of miRNAs involved in brain morphogenesis. MicroRNAs are abundant gene regulatory factors whose function in animal development and homeostasis is poorly understood. A new study reports the genetic elimination of miRNA function on a full genomic scale and identifies a subfamily of miRNAs involved in brain morphogenesis. One of the perhaps biggest surprises in molecular biology in the past few years has been the discovery of a large number of previously completely overlooked regulatory molecules, termed miRNAs. miRNAs, ~22 nt long RNA molecules, regulate the expression of target genes by binding to their 3′UTR [1Wightman B. Ha I. Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.Cell. 1993; 75: 855-862Abstract Full Text PDF PubMed Scopus (2872) Google Scholar] (Figure 1). They were first identified more than 10 years ago in the nematode Caenorhabditis elegans [2Lee R.C. Feinbaum R.L. Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (8662) Google Scholar], yet thought to be a worm specific oddity for the longest time. It was only the cloning of the second miRNA, let-7, that led the Ruvkun laboratory to note the conservation of miRNAs across phylogeny [3Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.Nature. 2000; 403: 901-906Crossref PubMed Scopus (3481) Google Scholar]. This in turn spurred intensive, genome-wide searches for miRNAs and current estimates of miRNA gene number range in the several hundreds for vertebrate genomes [4Bartel D.P. MicroRNAs. genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (26872) Google Scholar, 5Berezikov E. Guryev V. van de Belt J. Wienholds E. Plasterk R.H. Cuppen E. Phylogenetic shadowing and computational identification of human microRNA genes.Cell. 2005; 120: 21-24Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar]. But what is it exactly that miRNAs do and how do they do it? The only clear theme that has emerged over the past few years is that they generally appear to repress gene expression [1Wightman B. Ha I. Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.Cell. 1993; 75: 855-862Abstract Full Text PDF PubMed Scopus (2872) Google Scholar, 4Bartel D.P. MicroRNAs. genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (26872) Google Scholar]. But what cellular processes do miRNAs control? To date, there are still only four miRNAs —lin-4, let-7, bantam and lsy-6— whose physiological function has been elucidated in vivo and whose targets are known [2Lee R.C. Feinbaum R.L. Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (8662) Google Scholar, 3Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.Nature. 2000; 403: 901-906Crossref PubMed Scopus (3481) Google Scholar, 6Johnston R.J. Hobert O. A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans.Nature. 2003; 426: 845-849Crossref PubMed Scopus (591) Google Scholar, 7Stark A. Brennecke J. Russell R.B. Cohen S.M. Identification of Drosphila MicroRNA Targets.PLoS Biol. 2003; 1: 1-13Crossref Scopus (607) Google Scholar]. But speculation about the breadth of cellular processes in which animal miRNAs are involved have flourished over the past two years, mainly based on computational miRNA target prediction [8Lai E.C. Predicting and validating microRNA targets.Genome Biol. 2004; 5: 115Crossref PubMed Scopus (119) Google Scholar, 9Bartel D.P. Chen C.Z. Micromanagers of gene expression: the potentially widespread influence of metazoan miRNAs.Nat. Rev. Genet. 2004; 5: 396-400Crossref PubMed Scopus (1078) Google Scholar, 10Hobert O. Common logic of transcription factor and miRNA action.Trends Biochem. Sci. 2004; 29: 462-468Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar]. Yet, in contrast to plants, the usefulness of computational target prediction approaches has so far been limited in animals, which is illustrated by the striking lack of concordance of different target prediction algorithms. Nevertheless a common theme of all target predictions is that a large fraction of the genes in a given genome may be regulated by miRNAs. But how pervasive is miRNA function in reality? This is where a new study by Alex Schier’s lab [11Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson M.J. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; (epub ahead of print [DOI: 10.1126/Science. 1109020]): 000-000Google Scholar] has provided fundamentally important new insights. Rather than eliminating a single miRNA, Giraldez et al. [11Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson M.J. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; (epub ahead of print [DOI: 10.1126/Science. 1109020]): 000-000Google Scholar] eliminated all miRNAs by genetically removing the zebrafish gene coding for Dicer, an RNase required for miRNA processing [12Murchison E.P. Hannon G.J. miRNAs on the move: miRNA biogenesis and the RNAi machinery.Curr. Opin. Cell Biol. 2004; 16: 223-229Crossref PubMed Scopus (297) Google Scholar] (Figure 1) . A zebrafish Dicer mutant is not new per se; Plasterk and colleagues [13Wienholds E. Koudijs M.J. van Eeden F.J. Cuppen E. Plasterk R.H. The microRNA-producing enzyme Dicer1 is essential for zebrafish development.Nat. Genet. 2003; 35: 217-218Crossref PubMed Scopus (354) Google Scholar] had already reported the postembryonic lethality of Dicer knockout fish. However, their study was confounded by the fact that maternally supplied Dicer mRNA and/or protein from the heterozygous mothers of homozygous mutant embryos apparently allowed the generation of mature miRNAs during embryogenesis. This problem was now elegantly circumvented by using the germline replacement technique [14Ciruna B. Weidinger G. Knaut H. Thisse B. Thisse C. Raz E. Schier A.F. Production of maternal-zygotic mutant zebrafish by germ-line replacement.Proc. Natl. Acad. Sci. USA. 2002; 99: 14919-14924Crossref PubMed Scopus (159) Google Scholar], which allows the study of homozygous mutant embryos devoid of both maternal and zygotic Dicer function. The observations of Giraldez et al. [11Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson M.J. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; (epub ahead of print [DOI: 10.1126/Science. 1109020]): 000-000Google Scholar] on such maternal-zygotic Dicer mutant embryos are dramatic—not only because of the type of defect they observe, but also because of the type of defect they do not observe. Given the vast abundance of predicted miRNA target genes, including genes involved in signaling and transcriptional control, maternal-zygotic Dicer null mutants displayed surprisingly normal axis and pattern formation [9Bartel D.P. Chen C.Z. Micromanagers of gene expression: the potentially widespread influence of metazoan miRNAs.Nat. Rev. Genet. 2004; 5: 396-400Crossref PubMed Scopus (1078) Google Scholar]. Individual organs and multiple cell types were present and all anterior–posterior and dorsal–ventral patterning events examined do apparently not require miRNA function. Many of these initial patterning events are known to be under control of key signaling systems, such as Nodal, Hedgehog, Wnt, Notch, FGF, BMP and Retinoic acid [15Schier A.F. Axis formation and patterning in zebrafish.Curr. Opin. Genet. Dev. 2001; 11: 393-404Crossref PubMed Scopus (115) Google Scholar]. As many of these pathways were predicted by in silico approaches to be targeted by miRNAs, the absence of any defects in these systems upon global removal of miRNAs is striking. Given the negative nature of this result, the authors showed that miRNA processing is indeed globally defective in Dicer mutants using a representative sample of many miRNA species. However, it can formally not be excluded that trace amounts of miRNAs are still being produced, for example by an unknown RNase other than Dicer. In striking contrast to the lack of early patterning defects, Dicer mutants display severe defects in the morphogenesis of several distinct organ types [11Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson M.J. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; (epub ahead of print [DOI: 10.1126/Science. 1109020]): 000-000Google Scholar]. In the nervous system, neurulation was severely affected, brain ventricles did not form properly, subregions of the brains were not appropriately demarcated and neuron position and axon projections were disrupted. Gross defects were also observed in cell arrangements during gastrulation, cardiovascular morphogenesis and function as well as during somitogenesis. The third surprise of the paper came through an experiment that one would not have expected to work at all. Giraldez et al. [11Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson M.J. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; (epub ahead of print [DOI: 10.1126/Science. 1109020]): 000-000Google Scholar] re-supplied fish embryos with synthetic, mature miRNAs, which are beyond the step of requiring Dicer processing. Focusing on a set of miRNAs expressed at the relevant stages of development, the authors report that injections of a single subfamily (miR-430-a/b/c) of miRNAs can rescue the brain morphogenesis defects of Dicer mutants. However, some non-neuronal defects were either only partially rescued or not rescued at all, which hints at a tissue-specificity of miRNA function. Several aspects of these rescue experiments are notable. First, from a technical point of view they conclusively demonstrate that the observed effects of loss of Dicer are indeed caused by the lack of miRNA processing and not by other functions of Dicer, such as siRNA-dependent DNA methylation, histone modification and centromeric silencing [16Kanellopoulou C. Muljo S.A. Kung A.L. Ganesan S. Drapkin R. Jenuwein T. Livingston D.M. Rajewsky K. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.Genes Dev. 2005; 19: 489-501Crossref PubMed Scopus (1014) Google Scholar]. Second, it is as surprising as it is satisfying to see that many, very different aspects of brain morphogenesis are controlled by a single miRNA subfamily. This also makes miR-430-a/b/c the first vertebrate miRNAs with a firmly established role in development. It will be very intriguing to find the target genes of these miRNAs, particularly as some of the morphogenetic defects in Dicer null mutants, such as ventricle formation, are only poorly understood on a molecular level. Lastly, there is a notable oddity of the miR-430 subfamily — its three members come in a cluster of 90 copies. Clustered miRNA families have been known before but the sheer number of copies of miR-430 is unique. The functional relevance of this clustering— if there is one— remains mysterious. Naturally, we are now left with many new questions. Why are miRNAs abundantly employed in cell fate diversification rather than in early patterning? A general role of miRNAs in cell fate diversification can be extrapolated from several previous studies on individual miRNAs [6Johnston R.J. Hobert O. A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans.Nature. 2003; 426: 845-849Crossref PubMed Scopus (591) Google Scholar, 17Chang S. Johnston R.J. Frokjaer-Jensen C. Lockery S. Hobert O. MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode.Nature. 2004; 430: 785-789Crossref PubMed Scopus (279) Google Scholar, 18Chen C.Z. Li L. Lodish H.F. Bartel D.P. MicroRNAs modulate hematopoietic lineage differentiation.Science. 2004; 303: 83-86Crossref PubMed Scopus (2628) Google Scholar]. But why are they not employed in early patterning? Unfortunately, we currently understand too little about mechanisms of miRNA action to engage in anything but wild speculations. Perhaps there is something intrinsic about miRNA regulation that predestines miRNAs to fulfill a function in determining ‘stable’ gene expression programs in differentiating cell types. In one of the best characterized examples, the C. elegans miRNA lsy-6 is required to diversify and then lock two terminal differentiation programs into their stable end state [6Johnston R.J. Hobert O. A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans.Nature. 2003; 426: 845-849Crossref PubMed Scopus (591) Google Scholar]. In contrast, early patterning events are characterized by dynamic changes in gene expression. These regulatory dynamics require the activity of gene regulatory factors to be transient, plastic and/or reversible. Mechanistic features of miRNAs, such as their long half-life, may not allow them to participate in such dynamic processes. Another question is how general the observed zebrafish phenotype is. And here comes another surprise. In contrast to fish, knocking out Dicer in mice causes early embryonic death before axis formation [19Bernstein E. Kim S.Y. Carmell M.A. Murchison E.P. Alcorn H. Li M.Z. Mills A.A. Elledge S.J. Anderson K.V. Hannon G.J. Dicer is essential for mouse development.Nat. Genet. 2003; 35: 215-217Crossref PubMed Scopus (1473) Google Scholar]. Moreover, embryonic stem (ES) cells derived from Dicer null mutant mice do not form the three germ layers that can normally be found in ES-cell derived embryoid bodies [16Kanellopoulou C. Muljo S.A. Kung A.L. Ganesan S. Drapkin R. Jenuwein T. Livingston D.M. Rajewsky K. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.Genes Dev. 2005; 19: 489-501Crossref PubMed Scopus (1014) Google Scholar]. This may indicate a much broader role for miRNAs in early mouse embryos, but it is conceivable that this striking difference is ‘simply’explained by one or a few specific Dicer products that control a very early process such as cellular growth or viability. As any groundbreaking paper, the work of Giraldez et al. [11Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson M.J. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; (epub ahead of print [DOI: 10.1126/Science. 1109020]): 000-000Google Scholar] raises an abundance of new and exciting questions that will lead us further in our quest for understanding microRNA function.