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1.  OMIT: Dynamic, Semi-Automated Ontology Development for the microRNA Domain 
PLoS ONE  2014;9(7):e100855.
As a special class of short non-coding RNAs, microRNAs (a.k.a. miRNAs or miRs) have been reported to perform important roles in various biological processes by regulating respective target genes. However, significant barriers exist during biologists' conventional miR knowledge discovery. Emerging semantic technologies, which are based upon domain ontologies, can render critical assistance to this problem. Our previous research has investigated the construction of a miR ontology, named Ontology for MIcroRNA Target Prediction (OMIT), the very first of its kind that formally encodes miR domain knowledge. Although it is unavoidable to have a manual component contributed by domain experts when building ontologies, many challenges have been identified for a completely manual development process. The most significant issue is that a manual development process is very labor-intensive and thus extremely expensive. Therefore, we propose in this paper an innovative ontology development methodology. Our contributions can be summarized as: (i) We have continued the development and critical improvement of OMIT, solidly based on our previous research outcomes. (ii) We have explored effective and efficient algorithms with which the ontology development can be seamlessly combined with machine intelligence and be accomplished in a semi-automated manner, thus significantly reducing large amounts of human efforts. A set of experiments have been conducted to thoroughly evaluate our proposed methodology.
PMCID: PMC4099014  PMID: 25025130
2.  Burgeoning evidence indicates that microRNAs were initially formed from transposable element sequences 
Mobile Genetic Elements  2014;4:e29255.
MicroRNAs (miRNAs) constitute a recently discovered class of noncoding RNAs that play key roles in the regulation of gene expression. Despite being only ~20 nucleotides in length, these highly versatile molecules have been shown to play pivotal roles in development, basic cellular metabolism, apoptosis, and disease. While over 24,000 miRNAs have been characterized since they were first isolated in mammals in 2001, the functions of the majority of these miRNAs remain largely undescribed. That said, many now suggest that characterization of the relationships between miRNAs and transposable elements (TEs) can help elucidate miRNA functionality. Strikingly, over 20 publications have now reported the initial formation of thousands of miRNA loci from TE sequences. In this review we chronicle the findings of these reports, discuss the evolution of the field along with future directions, and examine how this information can be used to ascertain insights into miRNA transcriptional regulation and how it can be exploited to facilitate miRNA target prediction.
PMCID: PMC4091103  PMID: 25054081
microRNA; miR; miRNA; repetitive; retrotransposon; transposable; transposon
3.  Continuing analysis of microRNA origins 
Mobile Genetic Elements  2014;3(6):e27755.
MicroRNAs (miRs) are small noncoding RNAs that typically act as regulators of gene expression by base pairing with the 3′ UTR of messenger RNAs (mRNAs) and either repressing their translation or initiating degradation. As of this writing over 24,500 distinct miRs have been identified, but the functions of the vast majority of these remain undescribed. This paper represents a summary of our in depth analysis of the genomic origins of miR loci, detailing the formation of 1,213 of the 7,321 recently identified miRs and thereby bringing the total number of miR loci with defined molecular origin to 3,605. Interestingly, our analyses also identify evidence for a second, novel mechanism of miR locus generation through describing the formation of 273 miR loci from mutations to other forms of noncoding RNAs. Importantly, several independent investigations of the genomic origins of miR loci have now supported the hypothesis that miR hairpins are formed by the adjacent genomic insertion of two complementary transposable elements (TEs) into opposing strands. While our results agree that subsequent transcription over such TE interfaces leads to the formation of the majority of functional miR loci, we now also find evidence suggesting that a subset of miR loci were actually formed by an alternative mechanism—point mutations in other structurally complex, noncoding RNAs (e.g., tRNAs and snoRNAs).
PMCID: PMC3891635  PMID: 24475369
LINE; microRNA; miR; miRNA; noncoding RNA; repetitive; retrotransposon; SINE; transposable; transposon
4.  OrbId 
Mobile Genetic Elements  2012;2(4):184-192.
MicroRNAs coordinate networks of mRNAs, but predicting specific sites of interactions is complicated by the very few bases of complementarity needed for regulation. Although efforts to characterize the specific requirements for microRNA (miR) regulation have made some advances, no general model of target recognition has been widely accepted. In this work, we describe an entirely novel approach to miR target identification. The genomic events responsible for the creation of individual miR loci have now been described with many miRs now known to have been initially formed from transposable element (TE) sequences. In light of this, we propose that limiting miR target searches to transcripts containing a miR’s progenitor TE can facilitate accurate target identification. In this report we outline the methodology behind OrbId (Origin-based identification of microRNA targets). In stark contrast to the principal miR target algorithms (which rely heavily on target site conservation across species and are therefore most effective at predicting targets for older miRs), we find OrbId is particularly efficacious at predicting the mRNA targets of miRs formed more recently in evolutionary time. After defining the TE origins of > 200 human miRs, OrbId successfully generated likely target sets for 191 predominately primate-specific human miR loci. While only a handful of the loci examined were well enough conserved to have been previously evaluated by existing algorithms, we find ~80% of the targets for the oldest miR (miR-28) in our analysis contained within the principal Diana and TargetScan prediction sets. More importantly, four of the 15 OrbId miR-28 putative targets have been previously verified experimentally. In light of OrbId proving best-suited for predicting targets for more recently formed miRs, we suggest OrbId makes a logical complement to existing, conservation based, miR target algorithms.
PMCID: PMC3469430  PMID: 23087843
Alu; LINE; microRNA; miR; repetitive; target prediction; TE; transposable; UTR
5.  Comprehensive analysis of microRNA genomic loci identifies pervasive repetitive-element origins 
Mobile Genetic Elements  2011;1(1):8-17.
MicroRNAs (miRs) are small non-coding RNAs that generally function as negative regulators of target messenger RNAs (mRNAs) at the posttranscriptional level. MiRs bind to the 3′UTR of target mRNAs through complementary base pairing, resulting in target mRNA cleavage or translation repression. To date, over 15,000 distinct miRs have been identified in organisms ranging from viruses to man and interest in miR research continues to intensify. Of note, the most enlightening aspect of miR function—the mRNAs they target—continues to be elusive. Descriptions of the molecular origins of independent miR molecules currently support the hypothesis that miR hairpin generation is based on the adjacent insertion of two related transposable elements (TEs) at one genomic locus. Thus transcription across such TE interfaces establishes many, if not the majority of functional miRs. The implications of these findings are substantial for understanding how TEs confer increased genomic fitness, describing miR transcriptional regulations and making accurate miR target predictions. In this work, we have performed a comprehensive analysis of the genomic events responsible for the formation of all currently annotated miR loci. We find that the connection between miRs and transposable elements is more significant than previously appreciated, and more broadly, supports an important role for repetitive elements in miR origin, expression and regulatory network formation. Further, we demonstrate the utility of these findings in miR target prediction. Our results greatly expand the existing repertoire of defined miR origins, detailing the formation of 2,392 of 15,176 currently recognized miR genomic loci and supporting a mobile genetic element model for the genomic establishment of functional miRs.
PMCID: PMC3190270  PMID: 22016841
LINE; microRNA; miR; miRNA; repetitive; retroelement; SINE; transposable; transposon; UTR
6.  The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage 
Genome Biology  2013;14(3):R28.
We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.
Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.
Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.
PMCID: PMC4054807  PMID: 23537068
Amniote phylogeny; anoxia tolerance; chelonian; freeze tolerance; genomics; longevity; phylogenomics; physiology; turtle; evolutionary rates
7.  G-quadruplex recognition activities of E. Coli MutS 
BMC Molecular Biology  2012;13:23.
Guanine quadruplex (G4 DNA) is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA.
Here, we asked if E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements.
Taken together, our findings demonstrate that E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.
PMCID: PMC3437207  PMID: 22747774
DNA repair; G4; Quadruplex DNA; Mismatch repair; MutS
8.  Repression of human activation induced cytidine deaminase by miR-93 and miR-155 
BMC Cancer  2011;11:347.
Activation Induced cytidine Deaminase (AID) targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis.
The mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs) have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells.
We have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA.
Together, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located within the AID 3'UTR supports the recent suggestion that many miR regulations have been overlooked due to the prevalence of truncated 3'UTR annotations.
PMCID: PMC3163633  PMID: 21831295
AICDA; AID; CSR; hypermutation; microRNA; miR-93; miR-155; SHM; UTR; 3'UTR
9.  Histone H2A and H2B Are Monoubiquitinated at AID-Targeted Loci 
PLoS ONE  2010;5(7):e11641.
Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.
Methodology/Principal Findings
Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt's B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed VH and Sγ3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.
Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.
PMCID: PMC2905439  PMID: 20661291
10.  Adenosine deamination in human transcripts generates novel microRNA binding sites 
Human Molecular Genetics  2009;18(24):4801-4807.
Animals regulate gene expression at multiple levels, contributing to the complexity of the proteome. Among these regulatory events are post-transcriptional gene silencing, mediated by small non-coding RNAs (e.g. microRNAs), and adenosine-to-inosine (A-to-I) editing, generated by adenosine deaminases that act on double-stranded RNA (ADAR). Recent data suggest that these regulatory processes are connected at a fundamental level. A-to-I editing can affect Drosha processing or directly alter the microRNA (miRNA) sequences responsible for mRNA targeting. Here, we analyzed the previously reported adenosine deaminations occurring in human cDNAs, and asked if there was a relationship between A-to-I editing events in the mRNA 3′ untranslated regions (UTRs) and mRNA:miRNA binding. We find significant correlations between A-to-I editing and changes in miRNA complementarities. In all, over 3000 of the 12 723 distinct adenosine deaminations assessed were found to form 7-mer complementarities (known as seed matches) to a subset of human miRNAs. In 200 of the ESTs, we also noted editing within a specific 13 nucleotide motif. Strikingly, deamination of this motif simultaneously creates seed matches to three (otherwise unrelated) miRNAs. Our results suggest the creation of miRNA regulatory sites as a novel function for ADAR activity. Consequently, many miRNA target sites may only be identifiable through examining expressed sequences.
PMCID: PMC2778373  PMID: 19776031

Results 1-10 (10)