CiteULike: andersbj's library [261 articles]

The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

Sonia Guil — 2007-07-07T02:19:00-00:00

Nature Structural & Molecular Biology, Vol. 14, No. 7. (10 June 2007), pp. 591-596.

Useful ‘junk’: Alu RNAs in the human transcriptome

J Häsler — 2007-07-25T12:55:02-00:00

Cellular and Molecular Life Sciences (CMLS), Vol. 64, No. 14. (19 July 2007), pp. 1793-1800.

Abstract. Alu elements are the most abundant repetitive elements in the human genome; they have amplified by retrotransposition to reach the present number of more than one million copies. Alu elements can be transcribed in two different ways, by two independent polymerases. ‘Free Alu RNAs’ are transcribed by Pol III from their own promoter, while ‘embedded Alu RNAs’ are transcribed by Pol II as part of protein- and non-protein-coding RNAs. Recent studies have demonstrated that both free and embedded Alu RNAs play a major role in post transcriptional regulation of gene expression, for example by affecting protein translation, alternative splicing and mRNA stability. These discoveries illustrate how a part of the ‘junk DNA’ content of the human genome has been recruited to important functions in regulation of gene expression.

Brain Expressed microRNAs Implicated in Schizophrenia Etiology.

Thomas Hansen — 2007-09-13T18:12:29-00:00

PLoS ONE, Vol. 2, No. 9. (2007)

BACKGROUND: Protein encoding genes have long been the major targets for research in schizophrenia genetics. However, with the identification of regulatory microRNAs (miRNAs) as important in brain development and function, miRNAs genes have emerged as candidates for schizophrenia-associated genetic factors. Indeed, the growing understanding of the regulatory properties and pleiotropic effects that miRNA have on molecular and cellular mechanisms, suggests that alterations in the interactions between miRNAs and their mRNA targets may contribute to phenotypic variation. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the association between schizophrenia and genetic variants of miRNA genes associated with brain-expression using a case-control study design on three Scandinavian samples. Eighteen known SNPs within or near brain-expressed miRNAs in three samples (Danish, Swedish and Norwegian: 420/163/257 schizophrenia patients and 1006/177/293 control subjects), were analyzed. Subsequently, joint analysis of the three samples was performed on SNPs showing marginal association. Two SNPs rs17578796 and rs1700 in hsa-mir-206 (mir-206) and hsa-mit-198 (mir-198) showed nominal significant allelic association to schizophrenia in the Danish and Norwegian sample respectively (P = 0.0021 & p = 0.038), of which only rs17578796 was significant in the joint sample. In-silico analysis revealed that 8 of the 15 genes predicted to be regulated by both mir-206 and mir-198, are transcriptional targets or interaction partners of the JUN, ATF2 and TAF1 connected in a tight network. JUN and two of the miRNA targets (CCND2 and PTPN1) in the network have previously been associated with schizophrenia. CONCLUSIONS/SIGNIFICANCE: We found nominal association between brain-expressed miRNAs and schizophrenia for rs17578796 and rs1700 located in mir-206 and mir-198 respectively. These two miRNAs have a surprising large number (15) of targets in common, eight of which are also connected by the same transcription factors.

MOLECULAR BIOLOGY: miRNAs in Neurodegeneration

Sebastien Hebert — 2007-08-31T14:18:47-00:00

Science, Vol. 317, No. 5842. (31 August 2007), pp. 1179-1180.

10.1126/science.1148530

Computational analysis of microRNA-mediated antiviral defense in humans.

Yuka Watanabe — 2007-09-11T17:52:40-00:00

FEBS Lett (31 August 2007)

Recent studies have proposed the interesting perspective that viral gene expression is downregulated by host microRNAs (miRNAs), small non-coding RNAs well known as post-transcriptional gene regulators. We computationally predicted human miRNA target sites within 228 human-infecting and 348 invertebrate-infecting virus genomes, and we observed that human-infecting viruses were more likely than invertebrate-infecting ones to be targeted by human miRNAs. We listed 62 possible human miRNA-targeted viruses from 6 families, most of which consisted of single-stranded RNA viruses. These results suggest that miRNAs extensively mediate antiviral defenses in humans.

MicroRNA-34b and MicroRNA-34c Are Targets of p53 and Cooperate in Control of Cell Proliferation and Adhesion-Independent Growth.

David C Corney — 2007-09-09T16:39:35-00:00

Cancer Res (6 September 2007)

MicroRNAs (miRNA) are a recently discovered class of noncoding RNAs that negatively regulate gene expression. Recent evidence indicates that miRNAs may play an important role in cancer. However, the mechanism of their deregulation in neoplastic transformation has only begun to be understood. To elucidate the role of tumor suppressor p53 in regulation of miRNAs, we have analyzed changes in miRNA microarray expression profile immediately after conditional inactivation of p53 in primary mouse ovarian surface epithelium cells. Among the most significantly affected miRNAs were miR-34b and miR-34c, which were down-regulated 12-fold according to quantitative reverse transcription-PCR analysis. Computational promoter analysis of the mir-34b/mir-34c locus identified the presence of evolutionarily conserved p53 binding sites approximately 3 kb upstream of the miRNA coding sequence. Consistent with evolutionary conservation, mir-34b/mir-34c were also down-regulated in p53-null human ovarian carcinoma cells. Furthermore, as expected from p53 binding to the mir-34b/c promoter, doxorubicin treatment of wild-type, but not p53-deficient, cells resulted in an increase of mir-34b/mir-34c expression. Importantly, miR-34b and miR-34c cooperate in suppressing proliferation and soft-agar colony formation of neoplastic epithelial ovarian cells, in agreement with the partially overlapping spectrum of their predicted targets. Taken together, these results show the existence of a novel mechanism by which p53 suppresses such critical components of neoplastic growth as cell proliferation and adhesion-independent colony formation. [Cancer Res 2007;67(18):8433-8].

Cloning and Identification of Novel MicroRNAs from Rat Hippocampus.

X He — 2007-09-07T16:01:55-00:00

Acta Biochim Biophys Sin (Shanghai), Vol. 39, No. 9. (September 2007), pp. 708-714.

MicroRNAs (miRNAs) are small regulatory molecules post-transcriptionally suppressing mRNA activity. Many miRNAs in various organisms have been cloned but many unknown miRNAs remain to be identified. Here we describe the cloning of six new miRNAs from rat hippocampus. Among them, four were not found in the rat miRBase, but were identical to their human and/or mouse homolog, therefore they were designated as rno-miR-92b, rno-miR-146b, rno-let-7g, and rno-miR-551b. The other two were derived from the other arms of the known miRNA precursors of rno-miR-330 and rno-miR-384, and were not found in miRBase of all organisms. They were designated as rno-miR-330* and rno-miR-384*. The expression of these miRNAs was confirmed by RNA-tailing and primer-extension real-time reverse transcription-polymerase chain reaction. These six miRNAs were expressed at significantly higher levels in the hippocampus than in other tissues, including cerebral cortex, heart, liver, lung and kidney. miR-384* was 10 times more abundant than miR-384 in rat hippocampus, but little difference was found between miR-330* and miR-330 expression in the same tissue.

Ultraconserved Regions Encoding ncRNAs Are Altered in Human Leukemias and Carcinomas.

GA Calin — 2007-09-06T15:32:54-00:00

Cancer Cell, Vol. 12, No. 3. (September 2007), pp. 215-229.

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

MicroRNAs and Cell Cycle Regulation.

Michael Carleton — 2007-09-06T15:32:07-00:00

Cell Cycle, Vol. 6, No. 17. (26 June 2007)

MicroRNAs (microRNAs) are abundant, approximately 21-25 nucleotide (nt) non-coding RNAs that mediate sequence-specific, post-transcriptional repression of mRNA targets. Emerging evidence suggests that several microRNAs target transcripts that encode proteins directly or indirectly invovled in cell cycle progression and cellular proliferation. Moreover, alteration of microRNA levels can contribute to pathological conditions, including tumorigenesis, that are associated with loss of cell cycle control. In this review we highlight recent data linking microRNAs to mammalian cell cycle regulation. We describe how specific miRNAs function within pathways that control cell cycle checkpoints. We discuss emerging evidence that support the idea that some microRNA activity may be cell cycle dependent, and we outline how the coordinate regulation of microRNA targets mayinfluence cell cycle progression.

Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways

Patrice Dunoyer — 2007-06-28T22:35:23-00:00

Nature Genetics, Vol. 39, No. 7. (10 June 2007), pp. 848-856.

Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430.

Wen-Yee Choi — 2007-09-03T13:46:06-00:00

Science (30 August 2007)

MicroRNAs repress hundreds of target mRNAs, but the physiological roles of specific miRNA-mRNA interactions remain largely elusive. We report that zebrafish miR-430 dampens and balances the expression of the TGFss Nodal agonist squint and the TGFss Nodal antagonist lefty. To disrupt the interaction of specific miRNA-mRNA pairs, we developed target protector morpholinos complementary to miRNA binding sites in target mRNAs. Protection of squint or lefty mRNAs from miR-430 resulted in enhanced or reduced Nodal signaling, respectively. Simultaneous protection of squint and lefty or absence of miR-430 caused an imbalance and reduction in Nodal signaling. These findings establish an approach to analyze the in vivo roles of specific miRNA-mRNA pairs and reveal a requirement for miRNAs in dampening and balancing agonist/antagonist pairs.

A MicroRNA Feedback Circuit in Midbrain Dopamine Neurons.

J Kim — 2007-09-01T16:00:44-00:00

Science, Vol. 317, No. 5842. (31 August 2007), pp. 1220-1224.

MicroRNAs (miRNAs) are evolutionarily conserved, 18- to 25-nucleotide, non-protein coding transcripts that posttranscriptionally regulate gene expression during development. miRNAs also occur in postmitotic cells, such as neurons in the mammalian central nervous system, but their function is less well characterized. We investigated the role of miRNAs in mammalian midbrain dopaminergic neurons (DNs). We identified a miRNA, miR-133b, that is specifically expressed in midbrain DNs and is deficient in midbrain tissue from patients with Parkinson's disease. miR-133b regulates the maturation and function of midbrain DNs within a negative feedback circuit that includes the paired-like homeodomain transcription factor Pitx3. We propose a role for this feedback circuit in the fine-tuning of dopaminergic behaviors such as locomotion.

MicroRNA control of Nodal signalling

Graziano Martello — 2007-08-30T16:31:30-00:00

Nature (29 August 2007)

Tissue-dependent paired expression of miRNAs.

Seungil Ro — 2007-08-30T16:09:52-00:00

Nucleic Acids Res (28 August 2007)

It is believed that depending on the thermodynamic stability of the 5'-strand and the 3'-strand in the stem-loop structure of a precursor microRNA (pre-miRNA), cells preferentially select the less stable one (called the miRNA or guide strand) and destroy the other one (called the miRNA* or passenger strand). However, our expression profiling analyses revealed that both strands could be co-accumulated as miRNA pairs in some tissues while being subjected to strand selection in other tissues. Our target prediction and validation assays demonstrated that both strands of a miRNA pair could target equal numbers of genes and that both were able to suppress the expression of their target genes. Our finding not only suggests that the numbers of miRNAs and their targets are much greater than what we previously thought, but also implies that novel mechanisms are involved in the tissue-dependent miRNA biogenesis and target selection process.

Detection of a MicroRNA Signal in an In Vivo Expression Set of mRNAs.

T Liu — 2007-08-30T15:27:22-00:00

PLoS ONE, Vol. 2, No. 8. (2007)

BACKGROUND: microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of a miRNA to the target mRNA. As a result, translation of the target mRNA is inhibited and the mRNA may be destabilized. The inhibitory effects of miRNAs have been linked to diverse cellular processes including malignant proliferation, apoptosis, development, differentiation, and metabolic processes. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. METHODOLOGY/PRINCIPAL FINDINGS: RNA was extracted from 12 human primary brain tumor biopsies. These samples were used to determine genome-wide mRNA expression levels by microarray analysis and a miRNA profile by real-time reverse transcription PCR. Correlation coefficients were determined for all possible mRNA-miRNA pairs and the distribution of these correlations compared to the random distribution. An excess of high positive and negative correlation pairs were observed at the tails of these distributions. Most of these highest correlation pairs do not contain sufficiently complementary sequences to predict a target relationship; nor do they lie in physical proximity to each other. However, by examining pairs in which the significance of the correlation coefficients is modestly relaxed, negative correlations do tend to predict targets and positive correlations tend to predict physically proximate pairs. A subset of high correlation pairs were experimentally validated by over-expressing or suppressing a miRNA and measuring the correlated mRNAs. CONCLUSIONS/SIGNIFICANCE: Sufficient information exists within a set of tumor samples to detect endogenous correlations between miRNA and mRNA levels. Based on the validations the causal arrow for these correlations is likely to be directed from the miRNAs to the mRNAs. From these data sets, we inferred and validated a tumor suppression pathway linked to miR-181c.

MicroRNAs in Tumorigenesis: A Primer.

W Zhang — 2007-08-30T12:09:40-00:00

Am J Pathol, Vol. 171, No. 3. (September 2007), pp. 728-738.

MicroRNAs (miRNAs) are a family of 21- to 25-nucleotide, noncoding small RNAs that primarily function as gene regulators. It is surprising that these tiny molecules, so diverse and consequential in their biological functions, have been hidden for so many years. Thanks to their discovery, cancer research has found a new arena. Aided by innovative molecular techniques, the research of miRNAs in oncology has progressed rapidly in recent years. miRNA abnormalities are becoming an emerging theme in cancer research. Specific functions of miRNAs, many of which are relevant to cancer development, are becoming apparent. The value of miRNAs in cancer classification and prognostication is being explored, and new therapeutic strategies targeting miRNAs are being developed. Because there is great promise that miRNA research will provide breakthroughs in the understanding of cancer pathogenesis and development of new valuable prognostic markers, pathologists should be adequately informed of this rapidly progressing field. Here, we offer a review on the basics of miRNA biology and the emerging role of miRNA in cancer pathogenesis, classification, and prognostication, including highlights of the involvement of specific miRNAs in different tumor types.

microRNAs: A Safeguard against Turmoil?

Anthony Leung — 2007-08-24T09:57:39-00:00

Cell, Vol. 130, No. 4. (24 August 2007), pp. 581-585.

Emerging data suggest that microRNAs (miRNAs) are instrumental in a variety of stress responses in addition to their more recognized role in development. Surprisingly, miRNAs, which normally suppress expression of target transcripts, may become activators of expression during stress. This might be partially explained by new interactions of miRNA/Argonaute complexes with RNA-binding proteins that relocate from different subcellular compartments during stress.

Robust Machine Learning Algorithms Predict MicroRNA Genes and Targets.

Pål Sætrom — 2007-08-28T23:09:19-00:00

Methods Enzymol, Vol. 427C (2007), pp. 25-49.

MicroRNAs (miRNA) are nonprotein coding RNAs with the potential to regulate the gene expression of thousands of protein coding genes. Current estimates suggest the number of miRNA genes may be twice of what is currently known, and the mechanisms governing miRNA targeting remain elusive. Machine learning algorithms can be used to create classifiers that capture the characteristics of verified examples to determine whether genomic hairpins are similar to verified miRNA genes or if message 3'UTRs possess known target characteristics. Algorithms can never replace biological verifications, but should always be used to guide experimental design. This chapter focuses on potential problems that must be addressed when machine learning is used and follows a practical approach to demonstrate how support vector machines and genetic programming can predict miRNA genes and targets.

The Drosophila nerfin-1 mRNA requires multiple microRNAs to regulate its spatial and temporal translation dynamics in the developing nervous system.

Alexander Kuzin — 2007-08-24T18:53:18-00:00

Dev Biol (24 July 2007)

The mRNA encoding the Drosophila Zn-finger transcription factor Nerfin-1, required for CNS axon pathfinding events, is subject to post-transcriptional silencing. Although nerfin-1 mRNA is expressed in many neural precursor cells including all early delaminating CNS neuroblasts, the encoded Nerfin-1 protein is detected only in the nuclei of neural precursors that divide just once to generate neurons and then only transiently in nascent neurons. Using a nerfin-1 promoter-controlled reporter transgene, replacement of the nerfin-1 3' UTR with the viral SV-40 3' UTR releases the neuroblast translational block and prolongs reporter protein expression in neurons. Comparative genomics analysis reveals that the nerfin-1 mRNA 3' UTR contains multiple highly conserved sequence blocks that either harbor and/or overlap 21 predicted binding sites for 18 different microRNAs. To determine the functional significance of these microRNA-binding sites and less conserved microRNA target sites, we have studied their ability to block or limit the expression of reporter protein in nerfin-1-expressing cells during embryonic development. Our results indicate that no single microRNA is sufficient to fully inhibit protein expression but rather multiple microRNAs that target different binding sites are required to block ectopic protein expression in neural precursor cells and temporally restrict expression in neurons. Taken together, these results suggest that multiple microRNAs play a cooperative role in the post-transcriptional regulation of nerfin-1 mRNA, and the high degree of microRNA-binding site evolutionary conservation indicates that all members of the Drosophila genus employ a similar strategy to regulate the onset and extinction dynamics of Nerfin-1 expression.

miRNomics--The bioinformatic approach

Zhumur Ghosh — 2007-08-17T13:58:34-00:00

Biochemical and Biophysical Research Communications, Vol. In Press, Uncorrected Proof

MicroRNAs (miRNAs) are tiny genetic rheostats in plants, animals, and viruses, regulating the expression of messenger RNAs by targeting transcripts for cleavage or translational repression. Their regulatory impact is even more pervasive as a potential therapeutic tool. Since inception, computational methods have been an invaluable tool complementing experimental approaches. Here, we outline miRNA-bioinformatics highlighting the biological and therapeutic repertoire of miRNAs, in silico prediction of miRNA genes and their targets, along with a glimpse of the bioinformatic challenges that lie ahead.

MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system.

Marika Kapsimali — 2007-08-22T17:24:29-00:00

Genome Biology, Vol. 8 (21 August 2007), R173.

Conservation and evolution of miRNA regulatory programs in plant development.

Matthew R Willmann — 2007-08-23T07:12:43-00:00

Curr Opin Plant Biol (18 August 2007)

Over the past two years, microarray technologies, large-scale small RNA and whole genome sequencing projects, and data mining have provided a wealth of information about the spectrum of miRNAs and miRNA targets present in different plant species and the alga Chlamydomonas. Such studies have shown that a number of key miRNA regulatory modules for plant development are conserved throughout the plant kingdom, suggesting that these programs were crucial to the colonization of land. New genetic and biochemical studies of miRNA pathways in Arabidopsis, the spatiotemporal expression patterns of several conserved miRNAs and their targets, and the characterization of mutations in Arabidopsis and maize have begun to reveal the functions of these ancient miRNA-regulated developmental programs. In addition to these conserved miRNAs, there are many clade and species-specific miRNAs, which have evolved more recently and whose functions are currently unknown.

Crystal structure of human DGCR8 core.

Sun Young Sohn — 2007-08-21T14:22:55-00:00

Nat Struct Mol Biol (19 August 2007)

A complex of Drosha with DGCR8 (or its homolog Pasha) cleaves primary microRNA (pri-miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri-miRNAs. To clarify the molecular basis underlying recognition of pri-miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core (DGCR8S, residues 493-720). In the structure, the two double-stranded RNA-binding domains (dsRBDs) are arranged with pseudo two-fold symmetry and are tightly packed against the C-terminal helix. The H2 helix in each dsRBD is important for recognition of pri-miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri-miRNA in two possible orientations. We propose a model for DGCR8's recognition of pri-miRNA.

MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.

Margaret S Ebert — 2007-08-20T10:53:19-00:00

Nat Methods (12 August 2007)

MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

The roles of binding site arrangement and combinatorial targeting in microRNA repression of gene expression

Lawrence Hon — 2007-08-15T05:17:39-00:00

Genome Biology, Vol. 8 (14 August 2007), R166.

Raising the estimate of functional human sequences.

Michael Pheasant — 2007-08-13T08:26:55-00:00

Genome Res (9 August 2007)

While less than 1.5% of the mammalian genome encodes proteins, it is now evident that the vast majority is transcribed, mainly into non-protein-coding RNAs. This raises the question of what fraction of the genome is functional, i.e., composed of sequences that yield functional products, are required for the expression (regulation or processing) of these products, or are required for chromosome replication and maintenance. Many of the observed noncoding transcripts are differentially expressed, and, while most have not yet been studied, increasing numbers are being shown to be functional and/or trafficked to specific subcellular locations, as well as exhibit subtle evidence of selection. On the other hand, analyses of conservation patterns indicate that only approximately 5% (3%-8%) of the human genome is under purifying selection for functions common to mammals. However, these estimates rely on the assumption that reference sequences (usually ancient transposon-derived sequences) have evolved neutrally, which may not be the case, and if so would lead to an underestimate of the fraction of the genome under evolutionary constraint. These analyses also do not detect functional sequences that are evolving rapidly and/or have acquired lineage-specific functions. Indeed, many regulatory sequences and known functional noncoding RNAs, including many microRNAs, are not conserved over significant evolutionary distances, and recent evidence from the ENCODE project suggests that many functional elements show no detectable level of sequence constraint. Thus, it is likely that much more than 5% of the genome encodes functional information, and although the upper bound is unknown, it may be considerably higher than currently thought.

Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution.

K Tessmar-Raible — 2007-08-13T08:25:01-00:00

Cell, Vol. 129, No. 7. (29 June 2007), pp. 1389-1400.

Neurosecretory control centers form part of the forebrain in many animal phyla, including vertebrates, insects, and annelids. The evolutionary origin of these centers is largely unknown. To identify conserved, and thus phylogenetically ancient, components of neurosecretory brain centers, we characterize and compare neurons that express the prohormone vasotocin (vasopressin/oxytocin)-neurophysin in the developing forebrain of the annelid Platynereis dumerilii and of the zebrafish. These neurons express the same tissue-restricted microRNA, miR-7, and conserved, cell-type-specific combinations of transcription factors (nk2.1, rx, and otp) that specify their identity, as evidenced by the specific requirement of zebrafish rx3 for vasotocin-neurophysin expression. MiR-7 also labels another shared population of neurons containing RFamides. Since the vasotocinergic and RFamidergic neurons appear to be directly sensory in annelid and fish, we propose that cell types with dual sensory-neurosecretory properties were the starting point for the evolution of neurosecretory brain centers in Bilateria.

A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance.

Prasun J Mishra — 2007-08-11T04:17:04-00:00

Proc Natl Acad Sci U S A (8 August 2007)

MicroRNAs are predicted to regulate approximately 30% of all human genes by targeting sequences in their 3' UTR. Polymorphisms in 3' UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood. Here, we demonstrate that 829C-->T, a naturally occurring SNP, near the miR-24 binding site in the 3' UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance. miR-24 has a conserved binding site in DHFR 3' UTR. DHFR with WT and 3' UTR containing the 829C-->T mutation were expressed in DG44 cells that lack DHFR. Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3' UTR-expressing cells. Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells. Cells with the mutant 3' UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells. SNP-829C-->T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate. We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3' UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance.

Bayesian Inference of MicroRNA Targets from Sequence and Expression Data.

JC Huang — 2007-08-10T06:54:52-00:00

J Comput Biol, Vol. 14, No. 5. (June 2007), pp. 550-563.

MicroRNAs (miRNAs) regulate a large proportion of mammalian genes by hybridizing to targeted messenger RNAs (mRNAs) and down-regulating their translation into protein. Although much work has been done in the genome-wide computational prediction of miRNA genes and their target mRNAs, an open question is how to efficiently obtain functional miRNA targets from a large number of candidate miRNA targets predicted by existing computational algorithms. In this paper, we propose a novel Bayesian model and learning algorithm, GenMiR++ (Generative model for miRNA regulation), that accounts for patterns of gene expression using miRNA expression data and a set of candidate miRNA targets. A set of high-confidence functional miRNA targets are then obtained from the data using a Bayesian learning algorithm. Our model scores 467 high-confidence miRNA targets out of 1,770 targets obtained from TargetScanS in mouse at a false detection rate of 2.5%: several confirmed miRNA targets appear in our high-confidence set, such as the interactions between miR-92 and the signal transduction gene MAP2K4, as well as the relationship between miR-16 and BCL2, an anti-apoptotic gene which has been implicated in chronic lymphocytic leukemia. We present results on the robustness of our model showing that our learning algorithm is not sensitive to various perturbations of the data. Our high-confidence targets represent a significant increase in the number of miRNA targets and represent a starting point for a global understanding of gene regulation.

MicroRNA-21 Regulates Expression of the PTEN Tumor Suppressor Gene in Human Hepatocellular Cancer.

F Meng — 2007-08-08T14:48:28-00:00

Gastroenterology, Vol. 133, No. 2. (August 2007), pp. 647-658.

Background & Aims: microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression negatively. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNA to tumor biology has not been established. Methods: We evaluated the expression of miRNA in human hepatocellular cancer (HCC) by expression profiling, and defined a target gene and biologically functional effect of an up-regulated miRNA. Results: miR-21 was noted to be highly overexpressed in HCC tumors and cell lines in expression profiling studies using a miRNA microarray. Inhibition of miR-21 in cultured HCC cells increased expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, and decreased tumor cell proliferation, migration, and invasion. In contrast-enhanced miR-21 expression by transfection with precursor miR-21 increased tumor cell proliferation, migration, and invasion. Moreover, an increase in cell migration was observed in normal human hepatocytes transfected with precursor miR-21. PTEN was shown to be a direct target of miR-21, and to contribute to miR-21 effects on cell invasion. Modulation of miR-21 altered focal adhesion kinase phosphorylation and expression of matrix metalloproteases 2 and 9, both downstream mediators of PTEN involved in cell migration and invasion. Conclusions: Aberrant expression of miR-21 can contribute to HCC growth and spread by modulating PTEN expression and PTEN-dependent pathways involved in mediating phenotypic characteristics of cancer cells such as cell growth, migration, and invasion.

The MicroRNA miR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-mRNA Splicing

Eugene Makeyev — 2007-08-04T09:34:27-00:00

Molecular Cell, Vol. 27, No. 3. (3 August 2007), pp. 435-448.

Summary Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing.

Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development.

Wigard P Kloosterman — 2007-08-07T13:17:57-00:00

PLoS Biol, Vol. 5, No. 8. (24 July 2007)

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system.

M Wakiyama — 2007-08-03T15:41:53-00:00

Genes Dev, Vol. 21, No. 15. (1 August 2007), pp. 1857-1862.

MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Here we describe a recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5'-cap and 3'-poly(A) tail, resulting in translational repression.

A small microRNA makes a Bic difference

Howell Moffett — 2007-08-01T16:03:32-00:00

Genome Biology, Vol. 8 (31 July 2007), 221.

HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR

Zachary Klase — 2007-07-31T06:04:44-00:00

BMC Molecular Biology, Vol. 8 (30 July 2007), 63.

Drosophila microRNAs Are Sorted into Functionally Distinct Argonaute Complexes after Production by Dicer-1.

K Förstemann — 2007-07-31T16:24:49-00:00

Cell, Vol. 130, No. 2. (27 July 2007), pp. 287-297.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Here, we show that despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2.

Sorting of Drosophila Small Silencing RNAs.

Yukihide Tomari — 2007-07-31T16:24:26-00:00

Cell, Vol. 130, No. 2. (27 July 2007), pp. 299-308.

In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. Here, we report that siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates-the miRNA/miRNA( *) and siRNA duplexes-from which Argonaute proteins are loaded. We find that the Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA( *) duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures.

Target mimics modulate miRNAs

Daniel Chitwood — 2007-07-27T23:29:57-00:00

Nature Genetics, Vol. 39, No. 8., pp. 935-936.

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.

Daniela Castanotto — 2007-07-31T16:14:44-00:00

Nucleic Acids Res (26 July 2007)

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.

Cloning and characterization of microRNAs from Brassica napus.

Lei Wang — 2007-07-31T16:12:36-00:00

FEBS Lett (16 July 2007)

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus.

p53-Mediated Activation of miRNA34 Candidate Tumor-Suppressor Genes.

Guido T Bommer — 2007-07-28T16:22:09-00:00

Curr Biol (24 July 2007)

BACKGROUND: In response to varied cell stress signals, the p53 tumor-suppressor protein activates a multitude of genes encoding proteins with functions in cell-cycle control, DNA repair, senescence, and apoptosis. The role of p53 in transcription of other types of RNAs, such as microRNAs (miRNAs) is essentially unknown. RESULTS: Using gene-expression analyses, reporter gene assays, and chromatin-immunoprecipitation approaches, we present definitive evidence that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues. Using array-based approaches and algorithm predictions, we define genes likely to be directly regulated by miRNA34, with cell-cycle regulatory genes being the most prominent class. In addition, we provide functional evidence, obtained via antisense oligonucleotide transfection and the use of mouse embryonic stem cells with loss of miRNA34a function, that the BCL2 protein is regulated directly by miRNA34. Finally, we demonstrate that the expression of two miRNA34s is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLCs) and that the restoration of miRNA34 expression inhibits growth of NSCLC cells. CONCLUSIONS: Taken together, the data suggest the miRNA34s might be key effectors of p53 tumor-suppressor function, and their inactivation might contribute to certain cancers.

MicroRNA Inhibition of Translation Initiation in Vitro by Targeting the Cap-Binding Complex eIF4F.

Géraldine Mathonnet — 2007-07-28T16:20:28-00:00

Science (26 July 2007)

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or mRNA destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract, when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, subsequently consolidate mRNA silencing.

MicroRNA regulation of human protein protein interaction network.

Han Liang — 2007-07-28T15:48:12-00:00

RNA (24 July 2007)

Since the functional state of a protein-protein interaction network depends on gene expression, a fundamental question is what relationships exist between protein interaction network and gene regulation. In particular, microRNAs have recently emerged as a major class of post-transcriptional regulators that influences a large proportion of genes in higher eukaryotes. Here we show that protein connectivity in the human protein-protein interaction network is positively correlated with the number of microRNA target-site types in the 3' untranslated regions of the gene encoding the protein and that interacting proteins tend to share more microRNA target-site types than random pairs. Moreover, our results demonstrate that microRNA targeting propensity for genes in different biological processes can be largely explained by their protein connectivity. Finally, we show that for hub proteins, microRNA regulation complexity is negatively correlated with clustering coefficient, suggesting that microRNA regulation is more important to inter-modular hubs than to intramodular ones. Taken together, our study provides the first evidence for global correlation between microRNA repression and protein-protein interactions.

Target mimicry provides a new mechanism for regulation of microRNA activity.

José Manuel Franco-Zorrilla — 2007-07-24T20:11:33-00:00

Nat Genet (22 July 2007)

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (P(i)) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot P(i) content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of P(i) homeostasis, as demonstrated using artificial target mimics.

Global and Local Architecture of the Mammalian microRNA-Transcription Factor Regulatory Network.

Reut Shalgi — 2007-07-17T17:37:08-00:00

PLoS Comput Biol, Vol. 3, No. 7. (13 July 2007)

microRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. It is anticipated that, in combination with transcription factors (TFs), they span a regulatory network that controls thousands of mammalian genes. Here we set out to uncover local and global architectural features of the mammalian miR regulatory network. Using evolutionarily conserved potential binding sites of miRs in human targets, and conserved binding sites of TFs in promoters, we uncovered two regulation networks. The first depicts combinatorial interactions between pairs of miRs with many shared targets. The network reveals several levels of hierarchy, whereby a few miRs interact with many other lowly connected miR partners. We revealed hundreds of "target hubs" genes, each potentially subject to massive regulation by dozens of miRs. Interestingly, many of these target hub genes are transcription regulators and they are often related to various developmental processes. The second network consists of miR-TF pairs that coregulate large sets of common targets. We discovered that the network consists of several recurring motifs. Most notably, in a significant fraction of the miR-TF coregulators the TF appears to regulate the miR, or to be regulated by the miR, forming a diversity of feed-forward loops. Together these findings provide new insights on the architecture of the combined transcriptional-post transcriptional regulatory network.

Patterns of Known and Novel Small RNAs in Human Cervical Cancer.

WO Lui — 2007-07-10T16:20:12-00:00

Cancer Res, Vol. 67, No. 13. (1 July 2007), pp. 6031-6043.

Recent studies suggest that knowledge of differential expression of microRNAs (miRNA) in cancer may have substantial diagnostic and prognostic value. Here, we use a direct sequencing method to characterize the profiles of miRNAs and other small RNA segments for six human cervical carcinoma cell lines and five normal cervical samples. Of 166 miRNAs expressed in normal cervix and cancer cell lines, we observed significant expression variation of six miRNAs between the two groups. To further show the biological relevance of our findings, we examined the expression level of two significantly varying miRNAs in a panel of 29 matched pairs of human cervical cancer and normal cervical samples. Reduced expression of miR-143 and increased expression of miR-21 were reproducibly displayed in cancer samples, suggesting the potential value of these miRNAs as tumor markers. In addition to the known miRNAs, we found a number of novel miRNAs and an additional set of small RNAs that do not meet miRNA criteria. [Cancer Res 2007;67(13):6031-43].

MicroRNA Targeting Specificity in Mammals: Determinants beyond Seed Pairing.

Andrew Grimson — 2007-07-07T18:53:13-00:00

Mol Cell, Vol. 27, No. 1. (6 July 2007), pp. 91-105.

Mammalian microRNAs (miRNAs) pair to 3'UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are approximately 7 nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13-16, positioning within the 3'UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets.

Sequence and Expression Differences Underlie Functional Specialization of Arabidopsis MicroRNAs miR159 and miR319.

Javier F Palatnik — 2007-07-05T13:24:34-00:00

Dev Cell, Vol. 13, No. 1. (3 July 2007), pp. 115-125.

Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences.

Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs.

JL Rinn — 2007-07-04T08:22:29-00:00

Cell, Vol. 129, No. 7. (29 June 2007), pp. 1311-1323.

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing.

P Landgraf — 2007-07-04T08:21:49-00:00

Cell, Vol. 129, No. 7. (29 June 2007), pp. 1401-1414.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.