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1.  Most Caenorhabditis elegans microRNAs Are Individually Not Essential for Development or Viability 
PLoS Genetics  2007;3(12):e215.
MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs.
Author Summary
MicroRNAs (miRNAs) are tiny endogenous RNAs that regulate gene expression in plants and animals. Individual miRNAs have important roles in development, immunity, and cancer. Although the investigation of miRNA function is of great importance, to date few miRNAs have been studied in the intact organism because of a lack of mutants in which specific miRNAs have been inactivated. Here we describe a collection of loss-of-function mutants representing the majority of all known miRNA genes in the nematode Caenorhabditis elegans. This study identifies a new role for miRNAs in C. elegans and also demonstrates that most miRNAs are not essential for viability or development. Our findings suggest that many miRNAs act redundantly with other miRNAs or other pathways. We expect that this collection of miRNA mutants will become a widely used resource to further our understanding of the biology of miRNAs.
doi:10.1371/journal.pgen.0030215
PMCID: PMC2134938  PMID: 18085825
2.  Functional Analysis of Neuronal MicroRNAs in Caenorhabditis elegans Dauer Formation by Combinational Genetics and Neuronal miRISC Immunoprecipitation 
PLoS Genetics  2013;9(6):e1003592.
Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic “enhancer” approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.
Author Summary
MicroRNAs (miRNAs) are important in the regulation of gene expression and are present in many organisms. To identify specific biological processes that are regulated by miRNAs, we disturbed total miRNA function under a certain genetic background and searched for defects. Interestingly, we found a prominent developmental defect that was dependent on a mutation in another gene involved in regulating transcription in neurons. Thus, by compromising two different aspects of gene regulation, we were able to identify a specific biological function of miRNAs. By investigating this defect, we determined that neuronal miRNAs likely function to help modulate cyclic guanosine monophosphate signaling. We then took a systematic approach and identified many miRNAs and genes that are likely to be regulated by neuronal miRNAs, and in doing so, we found genes involved in the initial defect. Additionally, we found many other genes, and show that genes expressed in neurons seem to be more regulated by miRNAs than genes in the intestine. Through our study, we identify a biological function of neuronal miRNAs and provide data that will help in identifying other important, novel, and exciting roles of this important class of small RNAs.
doi:10.1371/journal.pgen.1003592
PMCID: PMC3688502  PMID: 23818874
3.  Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent 
Silence  2010;1:9.
Background
Antisense reagents can serve as efficient and versatile tools for studying gene function by inhibiting nucleic acids in vivo. Antisense reagents have particular utility for the experimental manipulation of the activity of microRNAs (miRNAs), which are involved in the regulation of diverse developmental and physiological pathways in animals. Even in traditional genetic systems, such as the nematode Caenorhabditis elegans, antisense reagents can provide experimental strategies complementary to mutational approaches. Presently no antisense reagents are available for inhibiting miRNAs in the nematode C. elegans.
Results
We have developed a new class of fluorescently labelled antisense reagents to inhibit miRNAs in developing worms. These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide. The dextran-conjugated antisense reagents can be conveniently introduced into the germline of adult hermaphrodites and are transmitted to their progeny, where they efficiently and specifically inhibit a targeted miRNA in different tissues, including the hypodermis, the vulva and the nervous system. We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.
Conclusion
This class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans. Combined with numerous mutants or reporter stains available, these reagents should provide a convenient approach to examine genetic interactions that involve miRNA, and may facilitate studying functions of miRNAs, especially ones whose deletion strains are difficult to generate.
See related research article: http://jbiol.com/content/9/3/20
doi:10.1186/1758-907X-1-9
PMCID: PMC2864223  PMID: 20359322
4.  CED-3 caspase acts with miRNAs to regulate non-apoptotic gene expression dynamics for robust development in C. elegans 
eLife  null;3:e04265.
Genetic redundancy and pleiotropism have limited the discovery of functions associated with miRNAs and other regulatory mechanisms. To overcome this, we performed an enhancer screen for developmental defects caused by compromising both global miRISC function and individual genes in Caenorhabditis elegans. Among 126 interactors with miRNAs, we surprisingly found the CED-3 caspase that has only been well studied for its role in promoting apoptosis, mostly through protein activation. We provide evidence for a non-apoptotic function of CED-3 caspase that regulates multiple developmental events through proteolytic inactivation. Specifically, LIN-14, LIN-28, and DISL-2 proteins are known miRNA targets, key regulators of developmental timing, and/or stem cell pluripotency factors involved in miRNA processing. We show CED-3 cleaves these proteins in vitro. We also show CED-3 down-regulates LIN-28 in vivo, possibly rendering it more susceptible to proteasomal degradation. This mechanism may critically contribute to the robustness of gene expression dynamics governing proper developmental control.
DOI: http://dx.doi.org/10.7554/eLife.04265.001
eLife digest
For an organism to develop from a single cell into a collection of many different, specialized cells, different genes must be switched on or off at particular times. However, some of these genes involved in development are ‘redundant’ and carry out the same or similar tasks. This acts like a backup system, so if one of the genes is unable to complete a task, the others can compensate and the organism will still develop correctly.
To produce a protein from a gene, the DNA sequence that makes up the gene is used as a template to create another molecule called messenger RNA. Genes can also be ‘silenced’—prevented from making proteins—by small molecules called microRNAs, which bind to messenger RNA molecules and mark them for destruction. MicroRNA molecules therefore play an important role in controlling development. However, as many microRNA molecules often work together, and as many genes are redundant, it can be difficult to discover the effects of specific microRNAs. It is also difficult to discover whether any other mechanisms work alongside the microRNAs to control development.
Weaver, Zabinsky et al. used mutant forms of the nematode worm Caenorhabditis elegans, in which microRNA gene regulation did not work correctly, to investigate the mechanisms that work alongside microRNAs to control development. Genes in these worms were silenced; those silenced genes that caused additional developmental defects were considered likely to work ‘redundantly’ in the same role as a microRNA molecule. This revealed over one hundred genes that were previously unknown to work with microRNA molecules.
Weaver, Zabinsky et al. focused on one of these genes, called ced-3. The CED-3 protein produced from this gene is known to execute programmed cell death, a carefully controlled process also known as apoptosis, but was not known to have other developmental functions. However, the worms with mutant forms of the ced-3 gene already have problems performing apoptosis but are otherwise relatively normal, so Weaver, Zabinsky et al. reasoned that the CED-3 protein must also have another role in development.
Further investigation revealed that ced-3 mutations most severely disrupt development when they are combined with mutations in one particular family of microRNAs. These microRNAs are particularly important for controlling both when cells specialize into a particular type of cell, and the timing of when certain stages of development happen. Experiments using purified proteins showed that CED-3 breaks down three proteins that are produced from genes controlled by this family of microRNA molecules, and one of these proteins was also broken down by CED-3 in experiments with mutant worms. Weaver, Zabinsky et al. therefore propose that CED-3 is part of a semi-redundant system that ensures the proteins are produced at the right level and at the right time even if the microRNAs insufficiently regulate them. This finding demonstrated both a specific role and specific targets for the CED-3 protein during development, entirely distinct from its role in apoptosis.
Although Weaver, Zabinsky et al. have identified a large number of genes that work alongside microRNAs to control development, these are only the genes that cause obvious developmental defects in healthy worms. Further experiments using similar techniques performed on worms under stress may reveal yet more such genes.
DOI: http://dx.doi.org/10.7554/eLife.04265.002
doi:10.7554/eLife.04265
PMCID: PMC4279084  PMID: 25432023
GW182; miRNA; caspase; DIS3L2; LIN28; heterochronic; C. elegans
5.  Multiple distinct small RNAs originate from the same microRNA precursors 
Genome Biology  2010;11(8):R81.
Background
MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes.
Results
We report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes.
Conclusions
We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.
doi:10.1186/gb-2010-11-8-r81
PMCID: PMC2945783  PMID: 20696037
6.  A Densely Interconnected Genome-Wide Network of MicroRNAs and Oncogenic Pathways Revealed Using Gene Expression Signatures 
PLoS Genetics  2011;7(12):e1002415.
MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA–pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes (“hubs”), most nodes in the miRNA–pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA–pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.
Author Summary
MicroRNAs (miRNAs) are naturally occurring small RNA molecules of ∼22 nucleotides that regulate gene expression. Recent studies have shown that miRNAs can behave as important components of cellular signaling pathways, as pathway regulators or pathway targets. Currently however, only a few miRNAs have been functionally linked to specific signaling pathways, raising the need for novel approaches to accelerate the identification of miRNA–pathway connections. Here, we show that gene expression signatures, previously used to reflect patterns of pathway activation, can also be used to represent miRNA activities. Using this approach, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Besides being the first study to conceptually demonstrate that expression signatures can act as surrogates of miRNA activity, our study provides a large database of candidate pathway-modulating miRNAs, which researchers interested in a particular pathway (e.g. Ras, Myc) are likely to find useful. Moreover, because this approach solely employs gene expression, it is immediately applicable to the thousands of microarray data sets currently available in the public domain.
doi:10.1371/journal.pgen.1002415
PMCID: PMC3240594  PMID: 22194702
7.  Pleiotropic constraints, expression level and the evolution of miRNA sequences 
Journal of molecular evolution  2013;77(0):206-220.
Post-transcriptional gene regulation mediated by microRNAs (miRNAs) plays critical roles during development by modulating gene expression and conferring robustness to stochastic errors. Phylogenetic analyses suggest that miRNA acquisition could play a role in phenotypic innovation. Moreover, miRNA-induced regulation strongly impacts genome evolution, increasing selective constraints on 3’UTRs, protein sequences and expression level divergence. Thus, it is essential to understand the factors governing sequence evolution for this important class of regulatory molecules. Investigation of the patterns of molecular evolution at miRNA loci have been limited in Caenorhabditis elegans because of the lack of a close outgroup. Instead, I used Caenorhabditis briggsae as the focus point of this study because of its close relationship to Caenorhabditis sp. 9. I also corroborated the patterns of sequence evolution in Caenorhabditis using published orthologous relationships among miRNAs in Drosophila. In nematodes and in flies, miRNA sequence divergence is not influenced by the genomic neighbourhood (i.e. intronic or intergenic) but is nevertheless affected by the genomic context because X-linked miRNAs evolve faster than autosomal miRNAs. However, this effect of chromosomal linkage can be explained by differential expression levels rather than a fast-X effect. The results presented here support a universal negative relationship between rates of molecular evolution and expression level, and suggest that mutations in highly expressed miRNAs are more likely to be deleterious because they potentially affect a larger number of target genes. Finally, I show that many single-family member miRNAs evolve faster than miRNAs from multigene families and have limited functional scope, suggesting that they are not strongly integrated in gene regulatory networks.
doi:10.1007/s00239-013-9588-6
PMCID: PMC3913099  PMID: 24100521
miRNA; expression level; molecular evolution; Caenorhabditis; Drosophila
8.  Using context-specific effect of miRNAs to identify functional associations between miRNAs and gene signatures 
BMC Bioinformatics  2013;14(Suppl 12):S1.
Background
MicroRNAs are a class of short regulatory RNAs that act as post-transcriptional fine-tune regulators of a large host of genes that play key roles in many cellular processes and signaling pathways. A useful step for understanding their functional role is characterizing their influence on the protein context of the targets. Using miRNA context-specific influence as a functional signature is promising to identify functional associations between miRNAs and other gene signatures, and thus advance our understanding of miRNA mode of action.
Results
In the current study we utilized the power of regularized regression models to construct functional associations between gene signatures. Genes that are influenced by miRNAs directly(computational miRNA target prediction) or indirectly (protein partners of direct targets) are defined as functional miRNA gene signature. The combined direct and indirect miRNA influence is defined as context-specific effects of miRNAs, and is used to identify regulatory effects of miRNAs on curated gene signatures. Elastic-net regression was used to build functional associations between context-specific effect of miRNAs and other gene signatures (disease, pathway signatures) by identifying miRNAs whose targets are enriched in gene lists. As a proof of concept, elastic-net regression was applied on lists of genes downregulated upon pre-miRNA transfection, and successfully identified the treated miRNA. This model was then extended to construct functional relationships between miRNAs and disease and pathway gene lists. Integrating context-specific effects of miRNAs on a protein network reveals more significant miRNA enrichment in prostate gene signatures compared to miRNA direct targets. The model identified novel list of miRNAs that are associated with prostate clinical variables.
Conclusions
Elastic-net regression is used as a model to construct functional associations between miRNA signatures and other gene signatures. Defining miRNA context-specific functional gene signature by integrating the downstream effect of miRNAs demonstrates better performance compared to the miRNA signature alone (direct targets). miRNA functional signatures can greatly facilitate miRNA research to uncover new functional associations between miRNAs and diseases, drugs or pathways.
doi:10.1186/1471-2105-14-S12-S1
PMCID: PMC3848857  PMID: 24267745
9.  microRNAs of parasitic helminths – Identification, characterization and potential as drug targets 
Graphical abstract
Highlights
•Importance of microRNAs in helminth post-transcriptional gene regulation is reviewed.•Increasing helminth miRNA data are available from deep sequencing.•Some miRNAs are helminth-specific, many are novel to each species.•miRNAs may regulate parasite and host gene expression.•Uptake of miRNA inhibitors and mimics is feasible for functional analysis.
microRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. They were first identified in the free-living nematode Caenorhabditis elegans, where the miRNAs lin-4 and let-7 were shown to be essential for regulating correct developmental progression. The sequence of let-7 was subsequently found to be conserved in higher organisms and changes in expression of let-7, as well as other miRNAs, are associated with certain cancers, indicating important regulatory roles. Some miRNAs have been shown to have essential functions, but the roles of many are currently unknown. With the increasing availability of genome sequence data, miRNAs have now been identified from a number of parasitic helminths, by deep sequencing of small RNA libraries and bioinformatic approaches. While some miRNAs are widely conserved in a range of organisms, others are helminth-specific and many are novel to each species. Here we review the potential roles of miRNAs in regulating helminth development, in interacting with the host environment and in development of drug resistance. Use of fluorescently-labeled small RNAs demonstrates uptake by parasites, at least in vitro. Therefore delivery of miRNA inhibitors or mimics has potential to alter miRNA activity, providing a useful tool for probing the roles of miRNAs and suggesting novel routes to therapeutics for parasite control.
doi:10.1016/j.ijpddr.2014.03.001
PMCID: PMC4095049  PMID: 25057458
microRNAs; Parasite; Helminth; Caenorhabditis elegans; Gene regulation; Therapeutic
10.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development 
PLoS Biology  2007;5(8):e203.
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.
Author Summary
The striking tissue-specific expression patterns of microRNAs (miRNAs) suggest that they play a role in tissue development. These small RNA molecules (∼22 bases in length) are processed from long primary transcripts (pri-miRNA) and regulate gene expression at the posttranscriptional level. There are hundreds of different miRNAs, many of which are strongly conserved. Vertebrate embryonic development is most easily studied in zebrafish, but genetically disrupting miRNA genes to see which miRNA does what is technically challenging. In this study, we interfere with miRNA function during the first few days of zebrafish embryonic development by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs). We show that morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA. We also used morpholinos to study the developmental effects of miRNA knockdown. Although we did not observe gross phenotypic defects for many miRNAs, we found that zebrafish miR-375 is essential for formation of the insulin-secreting pancreatic islet. Loss of miR-375 results in dispersed islet cells by 36 hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes.
The authors show that morpholinos can be used to knock down zebrafish miRNAs, revealing that miR-375 is important for vertebrate pancreas development.
doi:10.1371/journal.pbio.0050203
PMCID: PMC1925136  PMID: 17676975
11.  Evidence for Antisense Transcription Associated with MicroRNA Target mRNAs in Arabidopsis 
PLoS Genetics  2009;5(4):e1000457.
Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways.
Author Summary
Antisense transcription is a pervasive but poorly understood phenomenon in a wide variety of organisms. We have found evidence for a novel source of antisense transcription in Arabidopsis thaliana associated with miRNA targets via computational analyses of published whole-genome tiling microarray data, deep sequencing smRNA datasets, and from custom high-resolution (3 nt) tiling microarray analysis. Our data show increased antisense transcription for select miRNA targets in the hua enhancer1-1 (hen1-1), a smRNA methyltransferase mutant, and the suppressor of gene silencing3-14 (sgs3-14) mutant that affects post-transcriptional gene silencing and leaf development. Additional results suggest that miRNA targets and MIRNA genes are subject to the activities of both the miRNA and RNA silencing pathways in which HEN1 and SGS3 may represent associated nodes. The analysis of sense–antisense transcripts using high-resolution tiling microarrays and genetic mutants provides a precise and sensitive means to study epigenetic activities. Our method of mining expression data of plant miRNAs targets and smRNAs is potentially applicable to the identification of epigenetic targets in metazoans, where computational methods for prediction of miRNAs and their targets lack power because of sequence degeneracy, and to identify loci producing antisense transcripts by triggers other than miRNA-directed cleavage.
doi:10.1371/journal.pgen.1000457
PMCID: PMC2664332  PMID: 19381263
12.  The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages 
PLoS ONE  2008;3(8):e2997.
Background
MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species.
Methodology/Principal Findings
We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis.
Conclusions/Significance
Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.
doi:10.1371/journal.pone.0002997
PMCID: PMC2500172  PMID: 18714353
13.  Direct sequencing and expression analysis of a large number of miRNAs in Aedes aegypti and a multi-species survey of novel mosquito miRNAs 
BMC Genomics  2009;10:581.
Background
MicroRNAs (miRNAs) are a novel class of gene regulators whose biogenesis involves hairpin structures called precursor miRNAs, or pre-miRNAs. A pre-miRNA is processed to make a miRNA:miRNA* duplex, which is then separated to generate a mature miRNA and a miRNA*. The mature miRNAs play key regulatory roles during embryonic development as well as other cellular processes. They are also implicated in control of viral infection as well as innate immunity. Direct experimental evidence for mosquito miRNAs has been recently reported in anopheline mosquitoes based on small-scale cloning efforts.
Results
We obtained approximately 130, 000 small RNA sequences from the yellow fever mosquito, Aedes aegypti, by 454 sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. We also performed bioinformatics analysis on the Ae. aegypti genome assembly to identify evidence for additional miRNAs. The combination of these approaches uncovered 98 different pre-miRNAs in Ae. aegypti which could produce 86 distinct miRNAs. Thirteen miRNAs, including eight novel miRNAs identified in this study, are currently only found in mosquitoes. We also identified five potential revisions to previously annotated miRNAs at the miRNA termini, two cases of highly abundant miRNA* sequences, 14 miRNA clusters, and 17 cases where more than one pre-miRNA hairpin produces the same or highly similar mature miRNAs. A number of miRNAs showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these miRNAs. Northern blots also revealed several miRNAs that showed stage-specific expression. Detailed expression analysis of eight of the 13 mosquito-specific miRNAs in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. Four of the 13 miRNAs are specific to certain lineage(s) within mosquitoes.
Conclusion
This study provides the first systematic analysis of miRNAs in Ae. aegypti and offers a substantially expanded list of miRNAs for all mosquitoes. New insights were gained on the evolution of conserved and lineage-specific miRNAs in mosquitoes. The expression profiles of a few miRNAs suggest stage-specific functions and functions related to embryonic development or blood feeding. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases.
doi:10.1186/1471-2164-10-581
PMCID: PMC2797818  PMID: 19961592
14.  Characterizing the role of miRNAs within gene regulatory networks using integrative genomics techniques 
By integrating genotype information, microRNA transcript abundances and mRNA expression levels, Eric Schadt and colleagues provide insights into the genetic basis of microRNA gene expression and the role of microRNAs within the liver gene-regulatory network.
This article demonstrates how integrative genomics techniques can be used to investigate novel classes of RNA molecules. Moreover, it represents one of the first examinations of the genetic basis of variation in miRNA gene expression.Our results suggest that miRNA transcript abundances are under more complex regulation than previously observed for mRNA abundances.We also demonstrate that miRNAs typically exist as highly connected hub nodes and function as key sensors within the liver transcriptional network.Additionally, our results provide support for two key hypotheses—namely, that miRNAs can act cooperatively or redundantly to regulate a given pathway, and that miRNAs play a subtle role by dampening expression of their target gene through the use of feedback loops.
Since their discovery less than two decades ago, microRNAs (miRNAs) have repeatedly been shown to play a regulatory role in important biological processes. These small single-stranded molecules have been found to regulate multiple pathways—such as developmental timing in worms; fat metabolism in flies; and stress response in plants—and have been established as key regulatory molecules with potential widespread influence on both fundamental biology and various diseases. In the past decade, a new approach referred to by a number of names (‘integrative genomics', ‘systems genetics' or ‘genetical genomics') has shown increasing levels of success in elucidating the complex relationships found in gene regulatory networks. This approach leverages multiple layers of information (such as genotype, gene expression and phenotype) to infer causal associations that are then used for a number of different purposes, including identifying drivers of diseases and characterizing molecular networks. More importantly, many of the causal relationships that have been identified using this approach have been experimentally tested and verified. By integrating miRNA transcript abundances with messenger RNA (mRNA) expression data and genetic data, we have demonstrated how integrative genomics approaches can be used to characterize the global role played by miRNAs within complex gene regulatory networks. Overall, we investigated approximately 30% of the registered mouse miRNAs with a focus on liver networks. Our analysis reveals that miRNAs exist as highly connected hub nodes and function as key sensors within the gene regulatory network. Further comparisons between the regulatory loci contributing to the variation observed in miRNA and mRNA expression levels indicate that while miRNAs are controlled by more loci than have previously been observed for mRNAs, the contribution from each locus is on average smaller for miRNAs. We also provide evidence supporting two key hypotheses in the field: (i) miRNAs can act cooperatively or redundantly to regulate a given pathway; and (ii) miRNAs may regulate expression of their target gene through the use of feedback loops.
Integrative genomics and genetics approaches have proven to be a useful tool in elucidating the complex relationships often found in gene regulatory networks. More importantly, a number of studies have provided the necessary experimental evidence confirming the validity of the causal relationships inferred using such an approach. By integrating messenger RNA (mRNA) expression data with microRNA (miRNA) (i.e. small non-coding RNA with well-established regulatory roles in a myriad of biological processes) expression data, we show how integrative genomics approaches can be used to characterize the role played by approximately a third of registered mouse miRNAs within the context of a liver gene regulatory network. Our analysis reveals that the transcript abundances of miRNAs are subject to regulatory control by many more loci than previously observed for mRNA expression. Moreover, our results indicate that miRNAs exist as highly connected hub-nodes and function as key sensors within the transcriptional network. We also provide evidence supporting the hypothesis that miRNAs can act cooperatively or redundantly to regulate a given pathway and that miRNAs play a subtle role by dampening expression of their target gene through the use of feedback loops.
doi:10.1038/msb.2011.23
PMCID: PMC3130556  PMID: 21613979
causal associations; eQTL mapping; expression QTL; microRNA
15.  MicroRNA polymorphisms: the future of pharmacogenomics, molecular epidemiology and individualized medicine 
Pharmacogenomics  2009;10(3):399-416.
Referred to as the micromanagers of gene expression, microRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs. Polymorphisms in the miRNA pathway (miR-polymorphisms) are emerging as powerful tools to study the biology of a disease and have the potential to be used in disease prognosis and diagnosis. Detection of miR-polymorphisms holds promise in the field of miRNA pharmacogenomics, molecular epidemiology and for individualized medicine. MiRNA pharmacogenomics can be defined as the study of miRNAs and polymorphisms affecting miRNA function in order to predict drug behavior and to improve drug efficacy. Advancements in the miRNA field indicate the clear involvement of miRNAs and genetic variations within the miRNA pathway in the progression and prognosis of diseases such as cancer, neurological disorders, muscular hypertrophy, gastric mucosal atrophy, cardiovascular disease and Type II diabetes. Various algorithms are available to predict miRNA-target mRNA sites; however, it is advisable to use multiple algorithms to confirm the predictions. Polymorphisms that may potentially affect miRNA-mediated regulation of the cell can be present not only in the 3′-UTR of a miRNA target gene, but also in the genes involved in miRNA biogenesis and in pri-, pre- and mature-miRNA sequences. A polymorphism in processed miRNAs may affect expression of several genes and have serious consequences, whereas a polymorphism in miRNA target site, in the 3′-UTR of the target mRNA, may be more target and/or pathway specific. In this review, we for the first time suggest a classification of miRNA polymorphisms/mutations. We also describe the importance and implications of miR-polymorphisms in gene regulation, disease progression, pharmacogenomics and molecular epidemiology.
doi:10.2217/14622416.10.3.399
PMCID: PMC2705205  PMID: 19290790
classification; diagnosis; disease; drug resistance; epidemiology; epigenetics; microRNA; miRSNP; mutations; pharmacogenomics; polymorphism; prognosis; SNP
16.  Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel 
BMC Genomics  2012;13:4.
Background
MicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question.
Results
The small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes.
Conclusions
This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention.
doi:10.1186/1471-2164-13-4
PMCID: PMC3282659  PMID: 22216965
17.  A Negative Regulatory Loop between MicroRNA and Hox Gene Controls Posterior Identities in Caenorhabditis elegans 
PLoS Genetics  2010;6(9):e1001089.
MicroRNAs (miRNAs) have been found to regulate gene expression across eukaryotic species, but the function of most miRNA genes remains unknown. Here we describe how the analysis of the expression patterns of a well-conserved miRNA gene, mir-57, at cellular resolution for every minute during early development of Caenorhabditis elegans provided key insights in understanding its function. Remarkably, mir-57 expression shows strong positional bias but little tissue specificity, a pattern reminiscent of Hox gene function. Despite the minor defects produced by a loss of function mutation, overexpression of mir-57 causes dramatic posterior defects, which also mimic the phenotypes of mutant alleles of a posterior Hox gene, nob-1, an Abd homolog. More importantly, nob-1 expression is found in the same two posterior AB sublineages as those expressing mir-57 but with an earlier onset. Intriguingly, nob-1 functions as an activator for mir-57 expression; it is also a direct target of mir-57. In agreement with this, loss of mir-57 function partially rescues the nob-1 allele defects, indicating a negative feedback regulatory loop between the miRNA and Hox gene to provide positional cues. Given the conservation of the miRNA and Hox gene, the regulatory mechanism might be broadly used across species. The strategy used here to explore mir-57 function provides a path to dissect the regulatory relationship between genes.
Author Summary
miRNAs are small RNAs found in many multi-cellular species that inhibit gene expression. Many of them play important roles in cancer and cell fate determination, but the function of most miRNAs is uncertain. Using live cell imaging and automated expression analysis, we found a miRNA gene, mir-57, is expressed in a position rather than tissue dependent way. Hox genes also regulate cell fate patterning along anterior-posterior (a-p) axis across different tissues. By investigating interactions between genes of these classes expressed in mir-57 expressing cells, we demonstrated by both genetic analysis and gene expression assays that a negative feedback loop between a posterior Hox gene, nob-1, and mir-57 regulates posterior cell fate determination in C. elegans. On the one hand, the Hox gene is required for normal activation of mir-57 expression, and on the other, the Hox gene functions as a direct target of and is repressed by the miRNA. Given the conservation of the two genes, a negative feedback loop between Hox and miRNA genes might be broadly used across species to regulate cell fate along the a-p axis. Detailed expression analysis may provide a general way to dissect the regulatory role of miRNAs.
doi:10.1371/journal.pgen.1001089
PMCID: PMC2932687  PMID: 20824072
18.  In silico miRNA prediction in metazoan genomes: balancing between sensitivity and specificity 
BMC Genomics  2009;10:204.
Background
MicroRNAs (miRNAs), short ~21-nucleotide RNA molecules, play an important role in post-transcriptional regulation of gene expression. The number of known miRNA hairpins registered in the miRBase database is rapidly increasing, but recent reports suggest that many miRNAs with restricted temporal or tissue-specific expression remain undiscovered. Various strategies for in silico miRNA identification have been proposed to facilitate miRNA discovery. Notably support vector machine (SVM) methods have recently gained popularity. However, a drawback of these methods is that they do not provide insight into the biological properties of miRNA sequences.
Results
We here propose a new strategy for miRNA hairpin prediction in which the likelihood that a genomic hairpin is a true miRNA hairpin is evaluated based on statistical distributions of observed biological variation of properties (descriptors) of known miRNA hairpins. These distributions are transformed into a single and continuous outcome classifier called the L score. Using a dataset of known miRNA hairpins from the miRBase database and an exhaustive set of genomic hairpins identified in the genome of Caenorhabditis elegans, a subset of 18 most informative descriptors was selected after detailed analysis of correlation among and discriminative power of individual descriptors. We show that the majority of previously identified miRNA hairpins have high L scores, that the method outperforms miRNA prediction by threshold filtering and that it is more transparent than SVM classifiers.
Conclusion
The L score is applicable as a prediction classifier with high sensitivity for novel miRNA hairpins. The L-score approach can be used to rank and select interesting miRNA hairpin candidates for downstream experimental analysis when coupled to a genome-wide set of in silico-identified hairpins or to facilitate the analysis of large sets of putative miRNA hairpin loci obtained in deep-sequencing efforts of small RNAs. Moreover, the in-depth analyses of miRNA hairpins descriptors preceding and determining the L score outcome could be used as an extension to miRBase entries to help increase the reliability and biological relevance of the miRNA registry.
doi:10.1186/1471-2164-10-204
PMCID: PMC2688010  PMID: 19405940
19.  MIR846 and MIR842 comprise a cistronic MIRNA pair that is regulated by abscisic acid by alternative splicing in roots of Arabidopsis 
Plant molecular biology  2013;81(4-5):447-460.
MicroRNAs (miRNAs) are ~21-nucleotide long endogenous small RNAs that regulate gene expression through post-transcriptional or transcriptional gene silencing (PTGS/TGS) and/or translational inhibition. miRNAs can arise from the “exon” of a MIRNA gene, from an intron (e.g. mirtrons in animals), or from the antisense strand of a protein coding gene (natural antisense microRNAs, nat-miRNAs). Here we demonstrate that two functionally related miRNAs, miR842 and miR846, arise from the same transcription unit but from alternate splicing isoforms. miR846 is expressed only from Isoform1 while in Isoforms2 and -3, a part of pre-miR846 containing the miRNA* sequence is included in the intron. The splicing of the intron truncates the pre-MIRNA and disrupts the expression of the mature miR846.. We name this novel phenomenon splicing-regulated miRNA. Abscisic acid (ABA) is shown to mediate the alternative splicing event by reducing the functional Isoform1 and increasing the non-functional Isoform3, thus repressing the expression of miR846 concomitant with accumulation of an ABA-inducible target jacalin At5g28520 mRNA, whose cleavage was shown by modified 5′-RACE. This regulation shows the functional importance of splicing-regulated miRNA and suggests possible mechanisms for altered ABA response phenotypes of miRNA biogenesis mutants. A. lyrata-MIR842 and Aly-MIR846 have conserved genomic arrangements with A. thaliana and candidate target jacalins, similar primary transcript structures and intron processing, and better miRNA-miRNA* pairings, suggesting that the interactions between ABA, MIR842, MIR846 and jacalins are similar in A. lyrata. Together, splicing-regulated miRNAs, nat-miRNAs/inc-miRNAs and mirtrons illustrate the complexity of MIRNA genes, the importance of introns in the biogenesis and regulation of miRNAs, and raise questions about the processes and molecular mechanisms that drive MIRNA evolution.
doi:10.1007/s11103-013-0015-6
PMCID: PMC3581712  PMID: 23341152
alternative splicing; microRNA; root; abscisic acid; plant development; pri-miRNA
20.  Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans 
PLoS Genetics  2013;9(3):e1003353.
The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.
Author Summary
In the past decade, microRNAs (miRNAs) have become recognized as key regulators of gene expression in many biological pathways. These small, non-coding RNAs target specific protein-coding genes for repression. The specificity is mediated by partial base-pairing interactions between the 22 nucleotide miRNA and sequences in the target messenger RNA (mRNA). The use of imperfect base-pairing means that a single miRNA can regulate many different mRNAs, but it also means that identifying these targets is not straightforward. One of the first discovered miRNAs, let-7, generally promotes cellular differentiation pathways through a repertoire of targets that is yet to be fully described. Here we utilized molecular and genetic approaches to identify biologically relevant targets of the let-7 miRNA in Caenorhabditis elegans. Our analyses indicate that let-7 regulates a large cast of genes, both directly and indirectly. Loss of let-7 activity in C. elegans results in multiple developmental abnormalities and, ultimately, death. We uncovered new targets of let-7 that contribute to these phenotypes when they fail to be properly regulated. Given the highly conserved nature of let-7 from worms to humans, our studies highlight new genes and pathways potentially under let-7 regulation across species.
doi:10.1371/journal.pgen.1003353
PMCID: PMC3597506  PMID: 23516374
21.  Sequence Relationships among C. elegans, D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology 
PLoS ONE  2008;3(7):e2818.
microRNAs act in a prevalent and conserved post-transcriptional gene regulatory mechanism that impacts development, homeostasis and disease, yet biological functions for the vast majority of miRNAs remain unknown. Given the power of invertebrate genetics to promote rapid evaluation of miRNA function, recently expanded miRNA identifications (miRBase 10.1), and the importance of assessing potential functional redundancies within and between species, we evaluated miRNA sequence relationships by 5′ end match and overall homology criteria to compile a snapshot overview of miRNA families within the C. elegans and D. melanogaster genomes that includes their identified human counterparts. This compilation expands literature documentation of both the number of families and the number of family members, within and between nematode and fly models, and highlights sequences conserved between species pairs or among nematodes, flies and humans. Themes that emerge include the substantial potential for functional redundancy of miRNA sequences within species (84/139 C. elegans miRNAs and 70/152 D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes), and the striking extent to which miRNAs are conserved across species—over half (73/139) C. elegans miRNAs share sequence homology with miRNAs encoded also in both fly and human genomes. This summary analysis of mature miRNA sequence relationships provides a quickly accessible resource that should facilitate functional and evolutionary analyses of miRNAs and miRNA families.
doi:10.1371/journal.pone.0002818
PMCID: PMC2486268  PMID: 18665242
22.  Highly Dynamic and Sex-Specific Expression of microRNAs During Early ES Cell Differentiation 
PLoS Genetics  2009;5(8):e1000620.
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.
Author Summary
The discovery of the first microRNA (lin-4) in C. elegans in 1993 and the increasing realization that small RNAs are at the heart of many biological processes have led to a revolution in our thinking about development and disease. In animals, several hundred microRNAs (miRNAs) have been identified that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis, and cancer. Moreover, it has been shown that many miRNAs are characterized by highly specific spatial and temporal expression patterns supporting their role in such processes. However, the dynamics of small RNA patterns in male and female embryonic stem (ES) cells in the course of early differentiation has not been investigated so far. Our work represents the first study of this kind. Notably, we have identified new classes of miRNAs that show extremely defined temporal profiles during ES cell differentiation, as well as sex-specificity. Our results are of broad interest and importance because they raise the power of ES cells in defining the repertoire of small RNAs and their dynamics in mammals, and underline the importance of integrating miRNA expression patterns into the transcription factor networks and epigenomic maps defined in ES cells in order to provide a better understanding of the control of pluripotency and lineage commitment.
doi:10.1371/journal.pgen.1000620
PMCID: PMC2725319  PMID: 19714213
23.  Identification of Schistosoma mansoni microRNAs 
BMC Genomics  2011;12:47.
Background
MicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples.
Results
Homology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni.
Conclusion
Evidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites.
doi:10.1186/1471-2164-12-47
PMCID: PMC3034697  PMID: 21247453
24.  Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation 
Genome Biology  2004;5(3):R13.
Northern blot analysis of 119 previously reported microRNAs in adult organs from mouse and human identified a subset of brain-expressed miRNAs whose expression behavior is conserved in both mouse and human differentiating neurons, implicating these microRNAs in mammalian neuronal development or function
Background
The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18 to 25 nucleotides) with probable roles in the regulation of gene expression. In Caenorhabditis elegans, lin-4 and let-7 miRNAs control the timing of fate specification of neuronal and hypodermal cells during larval development. lin-4, let-7 and other miRNA genes are conserved in mammals, and their potential functions in mammalian development are under active study.
Results
In order to identify mammalian miRNAs that might function in development, we characterized the expression of 119 previously reported miRNAs in adult organs from mouse and human using northern blot analysis. Of these, 30 miRNAs were specifically expressed or greatly enriched in a particular organ (brain, lung, liver or skeletal muscle). This suggests organ- or tissue-specific functions for miRNAs. To test if any of the 66 brain-expressed miRNAs were present in neurons, embryonal carcinoma cells were treated with all-trans-retinoic acid to promote neuronal differentiation. A total of 19 brain-expressed miRNAs (including lin-4 and let-7 orthologs) were coordinately upregulated in both human and mouse embryonal carcinoma cells during neuronal differentiation. The mammalian ortholog of C. elegans lin-28, which is downregulated by lin-4 in worms via 3' untranslated region binding, was also repressed during neuronal differentiation of mammalian embryonal carcinoma cells. Mammalian lin-28 messenger RNAs contain conserved predicted binding sites in their 3' untranslated regions for neuron-expressed miR-125b (a lin-4 ortholog), let-7a, and miR-218.
Conclusions
The identification of a subset of brain-expressed miRNAs whose expression behavior is conserved in both mouse and human differentiating neurons implicates these miRNAs in mammalian neuronal development or function.
PMCID: PMC395763  PMID: 15003116
25.  The miR-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in Caenorhabditis elegans 
PLoS Genetics  2012;8(3):e1002536.
RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.
Author Summary
RNA interference (RNAi) has become a widely used approach for silencing genes of interest. This tool is possible because endogenous RNA silencing pathways exist broadly across organisms, including humans, worms, and plants. The general RNAi pathway utilizes small ∼21-nucleotide RNAs to target specific protein-coding genes through base-pairing interactions. Since RNAs from exogenous sources require some of the same factors as endogenous small RNAs to silence gene expression, there can be competition between the pathways. Thus, perturbations in the endogenous RNAi pathway can result in enhanced silencing efficiency by exogenous small RNAs. MicroRNAs (miRNAs) comprise another endogenous small RNA pathway, but their biogenesis and mechanism of gene silencing are distinct in many ways from RNAi pathways. Here we show that a family of miRNAs regulates the effectiveness of RNAi in Caenorhabditis elegans. Loss of mir-35-41 results in enhanced RNAi by exogenous RNAs and reduced silencing of endogenous RNAi targets. The embryonic miR-35-41 miRNAs regulate the sensitivity to RNAi through lin-35/Rb, a homolog of the human Retinoblastoma tumor suppressor gene previously shown to regulate RNAi effectiveness in C. elegans. Additionally, we show that this sensitivity can be passed on to the next generation of worms, demonstrating a far-reaching effect of the miR-35-41 miRNAs on gene regulation by other small RNA pathways.
doi:10.1371/journal.pgen.1002536
PMCID: PMC3297572  PMID: 22412382

Results 1-25 (923798)