MicroRNA Genomics and Biogenesis
Our lab was among those to first report the abundance of miRNAs in animals and the presence of miRNAs in plants. With help from collaborators, we went on to identify many of the miRNAs of model organisms (including C. elegans, fruit fly, Arabidopsis, and mice) and showed that humans have hundreds of miRNA genes. We also found miRNAs and another class of small regulatory RNAs known as piRNAs in the most deeply branching animals, implying that these two classes of regulatory RNAs have been available to shape gene expression since the beginning of multicellular animal life. Likewise, our results from deeply branching plants have revealed the presence of regulatory interactions involving miRNAs and other types of small RNAs in early land plants.
MicroRNAs are encoded by unusual genes whose primary transcripts form distinctive hairpin structures from which the miRNAs are processed. While analyzing data from high-throughput sequencing of small RNAs, we discovered types of miRNA precursors that bypass the specialized machinery that generate most miRNAs. In addition, we developed protocols to confidently identify the precursor hairpins that are recognized by the conventional machinery to give rise to miRNAs, and we have since turned our attention to how this recognition occurs, i.e., how the cellular machinery determines which of the many hairpin-containing transcripts enter the miRNA biogenesis pathway. Using combinatorial and biochemical approaches, we found primary sequence determinants, including a site for the SRp20 RNA-binding protein, that can help distinguish miRNA-containing transcripts from other types of hairpin-containing transcripts.
MicroRNA Regulatory Targets
The realization that genomes of animals and plants each have hundreds of miRNA genes raised the question of what all these tiny RNAs are doing. To address this question, we have developed increasingly reliable methods for predicting miRNA targets. In plants, we found that miRNAs have extensive pairing to their targets, and that evolutionarily conserved targets are mostly genes that play important roles during development. In animals, we have found a few cases of extensive pairing, but miRNAs usually recognize shorter sites (typically only 7 or 8 nucleotides in length) that match a short region of the miRNA containing the "seed" sequence. In collaboration with Christopher Burge (Massachusetts Institute of Technology), we showed that more than half of the human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. When we also consider nonconserved targeting, the fraction of human genes regulated by miRNAs grows even higher.
Although a 7-nucleotide site matching a miRNA often mediates some repression, it is not always sufficient, which indicates that other characteristics help specify targeting. For example, mRNA regions surrounding the sites can influence site accessibility and thus the efficacy of repression. Combining such features of site context, as well as other features that make some miRNAs more effective than others, we have constructed models that predict site performance, thereby providing important resources for choosing which of the many miRNA-target relationships are most promising for experimental follow-up. Our target predictions for mammals, zebrafish, flies, and nematodes can be viewed at www.TargetScan.org, with ranking of predictions available according to either their predicted efficacy or their preferential conservation.
MicroRNA Regulatory Effects and Biological Functions
To augment and inform our computational analyses of miRNA targeting, we have adapted high-throughput approaches to measure the effects of mammalian miRNAs on target mRNA levels, poly(A)-tail lengths, translational efficiency, and protein accumulation. Prior to these measurements, the prevailing view was that miRNAs mediate repression mostly through translational inhibition, with relatively little effect on target mRNA levels. However, our measurements showed that, although some translational repression can be detected, mammalian miRNAs predominantly act to decrease target mRNA levels. Messages from hundreds of genes are directly repressed by individual miRNAs, albeit each to a modest degree, indicating that for most interactions, miRNAs act as rheostats to adjust and optimize the mRNA output previously generated by transcription and pre-mRNA processing.
By disrupting the regulation of particular targets, several groups, including ours, have demonstrated the importance of miRNA-directed regulation during each stage of plant development. In addition, many groups have demonstrated the importance of miRNAs in animals. Indeed, our observation that most human genes are conserved targets of miRNAs indicates that it will be difficult to find a developmental process or disease that is not somehow influenced by miRNAs. We have contributed to the understanding of miRNA function during blood-cell, brain, and skeletal development, as well as cancer. For example, disrupting the miRNA regulation of the mouse Hmga2 oncogene enhances oncogenic transformation. Because many human tumors possess defective HMGA2 genes that lack the miRNA complementary sites, our work indicates that losing miRNA regulation of this oncogene contributes to some human cancers.
Other Small Regulatory RNAs
In addition to new miRNAs, our analysis of small RNAs found in animals, fungi, and plants uncovered other classes of endogenous small RNAs. These include the 21U- and 26G-RNAs of nematodes, the heterochromatic siRNAs (small interfering RNAs) of fungi, and what are now recognized as trans-acting siRNAs of plants. Experimental follow-up, mostly by other labs, has shown that, like miRNAs, these each play important gene-regulatory roles in RNA-silencing pathways that have emerged in their respective evolutionary lineages.
RNAi in Budding Yeast
The miRNA pathways of plants and animals appear to have evolved independently, both as elaborations of a core RNA-silencing pathway known as RNA interference (RNAi). During RNAi, the Dicer endonuclease processes long double-stranded RNA into siRNAs, which are then loaded into the Argonaute protein, where they direct the cleavage of mRNA targets. This pathway is present in most eukaryotes, where it plays important roles in defending against viruses and transposons, but it was initially thought to be absent in budding yeast. In collaboration with Gerry Fink (Whitehead Institute and MIT), we found that although RNAi has been lost in a recent ancestor of Saccharomyces cerevisiae, it is present in other budding yeasts, including Saccharomyces castellii (a close relative of S. cerevisiae) and Candida albicans (a common human pathogen).
The discovery of RNAi in budding yeast has opened many new opportunities for exploring the mechanism, biology, and evolution of the pathway. We found that introducing S. castellii Dicer and Argonaute into S. cerevisiae restores RNAi in this species. The reconstituted pathway strongly silences endogenous retrotransposons, which explains why it has been retained in S. castellii. On the other hand, endogenous dsRNA elements, known as Killer elements, are not retained in the RNAi-reconstituted S. cerevisiae strain, which renders these strains susceptible to the toxin of strains that have Killer. These results provide an explanation for why the presence of RNAi is so variable in the budding-yeast lineage: Retaining the pathway provides defense against transposons, whereas losing the pathway enables acquisition and retention of Killer.
In collaboration with Dinshaw Patel (Memorial Sloan-Kettering Cancer Center), whose lab solved the structures of the Dicer and Argonaute proteins, we have been studying the biochemical mechanism of RNAi in budding yeasts. We first determined the mechanism of the budding-yeast Dicer (Dcr1), which differs from other Dicer proteins, perhaps explaining why RNAi had gone undetected for so long in budding yeasts. We found that Dcr1 dimers bind cooperatively along the dsRNA substrate and cleave at precise intervals based on the distance between consecutive active sites. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, Dcr1 initiates processing in the interior and works outward. More recently, we have been studying the budding-yeast Argonaute (Ago1), which resembles Argonaute proteins of animals and plants. The structure of this protein together with its guide RNAs revealed how it positions these RNAs for target recognition and how a catalytic glutamine residue inserts into the active site to complete a previously unrecognized catalytic tetrad.
mRNA 3′ UTRs, Poly(A) Tails, and Developmental Transitions
MicroRNAs and other factors that mediate post-transcriptional gene regulation typically recognize elements in the 3′ untranslated regions (3′ UTRs) of mRNAs. Adding another dimension to this regulation is the use of alternative 3′ UTRs, which can either include or exclude the regulatory elements. To facilitate global analyses of these regulatory phenomena, we have developed a high-throughput method to accurately map the poly(A) sites of transcripts, which enables us to identify the ends of mRNAs and quantify the use of alternative isoforms in different cells, tissues or developmental stages. We have applied this method to expand and correct the mRNA annotations of C. elegans, zebrafish, mice, and humans. These studies have substantially improved our prediction of miRNA targets in each of these species and have revealed the regulatory consequences of alternative 3′ UTRs in mammalian cells.
In animals, maternal gene products deposited into eggs regulate embryonic development before the zygotic genome is activated. In plants, an analogous period of prolonged maternal control over embryogenesis was also thought to occur. Overturning this idea, we showed that the vast majority of Arabidopsis mRNAs are produced in near-equal amounts from both maternal and paternal alleles, even during the initial stages of embryogenesis.
In animals, we recently found a transition in translational control that follows the maternal-to-zygotic shift in transcriptional control. Key to this discovery was our development of a high-throughput method for measuring the lengths of poly(A) tails found at the ends of most eukaryotic mRNAs. When using this method to measure tail lengths of millions of individual mRNAs isolated from early zebrafish and frog embryos, we found a very strong correlation between tail length and translational efficiency, as would be expected if mRNAs with longer tails were more efficiently translated. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, which indicated a previously unrecognized developmental switch in the nature of translational control.
Long Non-coding RNAs
In humans and other mammals, thousands of genomic loci produce long intervening non-coding RNAs (lincRNAs), which resemble mRNAs yet do not encode proteins. To better understand the evolution of lincRNAs, we identified lincRNAs in zebrafish and C. elegans. Although our set of zebrafish lincRNAs share many characteristics with mammalian lincRNAs, only ~30 have detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Nonetheless, other lincRNAs are transcribed from conserved genomic locations despite our inability to detect sequence conservation.
To investigate the functions of these enigmatic RNAs, we examined two lincRNAs that each has a short region of deep sequence conservation. Antisense reagents targeting these conserved regions cause defects in embryonic development. Reagents targeting splice sites cause the same defects and are rescued by adding either the mature lincRNA or its human or mouse ortholog. Thus, lincRNAs can play crucial roles during embryonic development with conserved functionality despite limited sequence conservation. Interestingly, one of these lincRNAs has conserved pairing to a miRNA, which is required for its function in embryonic development.
Subtelny, A.O., S.W. Eichhorn, G.R. Chen, H. Sive and D.P. Bartel. 2014. Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature, in press.
Ulitsky, I. and D.P. Bartel. 2013. lincRNAs: Genomics, Evolution, and Mechanisms. Cell 154:26-46.
Auyeung, V.C., I. Ulitsky, S.E. McGeary and D.P. Bartel. 2013. Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing. Cell 152:844-858.
Nakanishi K, D.E. Weinberg, D.P. Bartel and D.J. Patel. 2012. Structure of yeast Argonaute with guide RNA. Nature 486:368-374.
Nodine, M.D. and D.P. Bartel. 2012. Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos. Nature 482:94-97.
Ulitsky, I, A. Shkumatava, C.H. Jan, H. Sive and D.P. Bartel. 2011. Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147:1537-1550.
Drinnenberg, I.A., G.R. Fink and D.P. Bartel. 2011. Compatibility with killer explains the rise of RNAi-deficient fungi. Science 333:1592.
Weinberg, D.E., K. Nakanishi, D.J. Patel and D.P. Bartel. 2011. The inside-out mechanism of Dicers from budding yeasts. Cell 146:262-276.
Jan, C.H., R.C. Friedman, J.G. Ruby and D.P. Bartel. 2011. Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs. Nature 469:97-101.
Guo, H., N.T. Ingolia, J.S. Weissman and D.P. Bartel. 2010. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466:835-840.
Shechner, D.M., R.A. Grant, S.C. Bagby, Y. Koldobskaya, J.A. Piccirilli and D.P. Bartel. 2009. Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326:1271-1275.
Drinnenberg, I.A., D.E. Weinberg, K.T. Xie, J.P. Mower, K.H. Wolfe, G.R. Fink and D.P. Bartel. 2009. RNAi in budding yeast. Science 326:544-550.
Mayr, C. and D.P. Bartel. 2009. Widespread shortening of 3′UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 38:673-684.
Bartel, D.P. 2009. MicroRNAs: Target recognition and regulatory functions. Cell 136:215-233.
Friedman, R.C., K.K. Farh, C.B. Burge and D.P. Bartel. 2009. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19:92-105.