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Small Regulatory RNAs
Summary: David Bartel explores the regulation of gene expression by small RNAs found in animal and plant cells.
We study small RNAs that regulate gene expression. Our main focus is on microRNAs (miRNAs), which are ~22-nt RNAs that specify gene repression by base-pairing to messages of protein-coding genes in plant and animal cells. Our lab is uncovering both the widespread influence of miRNAs on metazoan gene expression and the roles that miRNAs play during growth and development of plants and animals. For example, our work indicates that more than a third of human protein-coding genes are conserved regulatory targets of miRNAs and that the miRNA regulation of one of these genes is important for preventing human cancers.
Genomics of MicroRNAs
Our lab was among those that discovered the abundance of miRNAs. These tiny endogenous regulatory RNAs are encoded by unusual genes whose primary transcripts form distinctive hairpin structures from which the miRNAs are processed. Each hairpin is processed to generate the mature ~22-nt miRNA, which is then incorporated into a silencing complex, where it can specify post-transcriptional gene repression by base-pairing to messages of protein-coding genes.
We previously developed cloning and computational strategies for miRNA gene discovery in animals and plants and found, for example, that humans have hundreds of miRNA genes. We have also shown that many miRNAs are present at levels of more than 1,000 molecules per cell, with some exceeding 50,000 molecules per cell, indicating that the miRNAs, together with their associated proteins, are among the more abundant ribonucleoprotein complexes found in animal cells. Furthermore, many of the miRNAs from nematodes are related to those found in humans, suggesting important functions since early metazoan evolution. Nonetheless, some miRNAs, particularly those that are expressed in only a few cells and are less extensively conserved, were not identified by earlier cloning and computational efforts.
To explore more thoroughly the genomics of miRNAs and other endogenous silencing RNAs, we are using high-throughput sequencing methods to obtain millions of small RNA–sequencing reads from model plants and animals. Analyses of Physcomitrella (moss) and Arabidopsis sequences have revealed a layer of miRNA-based control involving miRNAs that are more diverse and evolutionarily fluid than those found previously. In plants we also find that dual miRNA complementary sites trigger the production of double-stranded RNA and endogenous siRNAs—an observation that provides insights into the recognition of aberrant RNA by the RNA-silencing machinery. Analysis of nematode and fly sequences has also revealed additional miRNAs, including some that had been missed earlier because their initial processing involves splicing rather than the specialized machinery that generates canonical miRNAs.
Other Small Regulatory RNAs
In addition to miRNAs and endogenous siRNAs, our high-throughput data have revealed previously unknown types of RNAs. Recently identified classes of small RNAs in animals include Piwi-interacting RNAs (piRNAs) and 21U-RNAs. The piRNAs (found in collaboration with Robert Kingston's lab [Massachusetts General Hospital, Boston] and also by several other labs) are ~29-nt RNAs that are likely involved in mammalian sperm development. The 21U-RNAs are an intriguing class of small RNAs found in worms. Precisely 21 nt long, they begin with a uridine but are diverse in their remaining 20 nt. They originate from >12,000 genomic loci dispersed in two broad regions of one chromosome—primarily between protein-coding genes or within their introns. These loci share a large upstream motif that is conserved in other nematodes, presumably because of its importance for producing this class of small RNAs.
Regulatory Targets of MicroRNAs
The discovery of hundreds of miRNA genes immediately raised the question of what all these tiny RNAs are doing. To address this question, we have developed methods of predicting miRNA targets without bringing in too many false-positive predictions. In plants, the miRNAs have extensive pairing to their targets, and the evolutionarily conserved targets are mostly genes that play important roles during development. In animals, the miRNAs usually recognize shorter sites (typically 7 or 8 nt in length) that match a short region of the miRNA containing the "seed" sequence. Our mammalian predictions, obtained in collaboration with Christopher Burge (Massachusetts Institute of Technology), can be viewed at www.TargetScan.org.
Animal miRNAs have a great abundance and diversity of targets, with more than one-third of human genes under selective pressure to maintain pairing to miRNAs. When nonconserved targeting is considered, the fraction of human genes regulated by miRNAs grows even higher. Experiments using reporter assays and mRNA expression arrays provide additional evidence that miRNAs have a widespread influence on both the expression and evolution of mammalian protein-coding genes. For example, mRNAs preferentially expressed in the same tissue as a highly expressed miRNA are strongly depleted in 7mer matches to that miRNA. Presumably this is because these messages have important roles in that tissue and during the course of evolution they have avoided acquiring sites for coexpressed miRNAs that would compromise their function. This selective avoidance of 7mer matches to miRNAs is compelling evidence that 7mer sites are often sufficient for repression in animals.
Although a 7mer site matching a miRNA is often sufficient for mediating some repression, it is not always sufficient, indicating that other characteristics help specify targeting. Using both computational and experimental approaches, we uncovered five general features of site context that boost site efficacy. Combining these features, we constructed a model of target recognition that successfully predicts site performance, providing an important resource for choosing which of the many miRNA-target relationships are most promising for experimental follow-up. Because our approach distinguishes effective from ineffective sites without recourse to evolutionary conservation, it also identifies effective nonconserved sites and siRNA off-targets.
Biological Functions of MicroRNAs
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. Our efforts, often in collaboration with Bonnie Bartel's lab (Rice University), have focused on the repression of the messages of NAC-domain transcription factors, auxin-response transcription factors, HD-Zip transcription factors, and ARGONAUTE1, a protein crucial for plant miRNA function. With regard to miRNA function in mammals, we showed that miR-196 directs the cleavage of HoxB8 mRNA during mouse embryonic development and also appears to repress paralogous Hox genes. More recently, we worked with Michael Hemann (Massachusetts Institute of Technology) to show that disrupting the miRNA regulation of the 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 human cancers.
RNA-Catalyzed RNA Polymerization
The RNA world hypothesis states that early life forms lacked protein enzymes and depended instead on enzymes composed of RNA. This hypothesis relies on the premise that some RNA sequences can catalyze RNA replication. In support of this idea, we have created an RNA molecule that catalyzes the type of polymerization reaction needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides—more than a complete turn of an RNA helix. Its polymerization is quite accurate, and most importantly, it is general in terms of the sequence and length of the primer and template RNAs, as would be needed for self-replication and evolution. We are examining the processivity and other catalytic and structural features of this ribozyme and its predecessors.
Our work on miRNAs and ribozymes is supported in part by grants from the National Institutes of Health.
Last updated: January 14, 2008
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