miR Loci Research Articles

Relationship of microRNA locus with type 2 diabetes mellitus: a case–control study

Type 2 diabetes mellitus (T2DM) is considered as a metabolic disease with hyperglycemia. Accumulating investigations have explored the important role of hereditary factors for T2DM occurrence. Some functional microRNA (miR) polymorphisms may affect their interactions with target mRNAs and result in an aberrant expression. Thus, miR variants might be considered as a biomarker of the susceptibility of T2DM. In this study, we recruited 502 T2DM cases and 782 healthy subjects. We selected miR-146a rs2910164 C>G, miR-196a2 rs11614913 T>C and miR-499 rs3746444 A>G loci and carried out an investigation to identify whether these miR loci could influence T2DM occurrence. In this investigation, a Bonferroni correction was harnessed. After adjustment, we found that rs2910164 SNP was a protective factor for T2DM (GG vs CC/CG: adjusted P = 0.010), especially in never drinking (GG vs CC/CG: adjusted P = 0.001) and BMI ≥24 kg/m2 (GG vs CC/CG: adjusted P = 0.002) subgroups. We also identified that rs11614913 SNP was a protective factor for T2DM in smoking subjects (CC/TC vs TT: adjusted P = 0.002). When we analyzed an interaction of SNP–SNP with the susceptibility tof T2DM, rs11614913/rs3746444, rs2910164/rs3746444 and rs11614913/rs2910164 combinations were not associated with the risk of T2DM. In summary, this study highlights that rs2910164 SNP decreases the susceptibility of T2DM, especially in BMI ≥24 kg/m2 and never drinking subgroups. In addition, we also identify that rs11614913 C allele decreases the susceptibility of T2DM significantly in smoking subgroup.

Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Levels

RNA binding proteins (RBPs) interact with primary, precursor, and mature microRNAs (miRs) to influence mature miR levels, which in turn affect critical aspects of human development and disease. To understand how RBPs contribute to miR biogenesis, we analyzed human enhanced UV crosslinking followed by immunoprecipitation (eCLIP) datasets for 126 RBPs to discover miR-encoding genomic loci that are statistically enriched for RBP binding. We find that 92% of RBPs interact directly with at least one miR locus, and that some interactions are cell line specific despite expression of the miR locus in both cell lines evaluated. We validated that ILF3 and BUD13 directly interact with and stabilize miR-144 and that BUD13 suppresses mir-210 processing to the mature species. We also observed that DDX3X regulates primary miR-20a, while LARP4 stabilizes precursor mir-210. Our approach to identifying regulators of miR loci can be applied to any user-defined RNA annotation, thereby guiding the discovery of uncharacterized regulators of RNA processing.

A genome-wide analysis of the RNA-guided silencing pathway in coffee reveals insights into its regulatory mechanisms.

microRNAs (miRNAs) are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs) are derived from double-stranded RNA (dsRNA) or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL) in processing the smallRNAs (sRNAs) and ARGONAUTE (AGO) as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR), Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated), and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA) pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop.

Landscape of Fluid Sets of Hairpin-Derived 21-/24-nt-Long Small RNAs at Seed Set Uncovers Special Epigenetic Features in Picea glauca.

Conifers’ exceptionally large genome (20–30 Gb) is scattered with 60% retrotransposon (RT) components and we have little knowledge on their origin and evolutionary implications. RTs may impede the expression of flanking genes and provide sources of the formation of novel small RNA (sRNAs) populations to constrain events of transposon (TE) proliferation/transposition. Here we show a declining expression of 24-nt-long sRNAs and low expression levels of their key processing gene, pgRTL2 (RNASE THREE LIKE 2) at seed set in Picea glauca. The sRNAs in 24-nt size class are significantly less enriched in type and read number than 21-nt sRNAs and have not been documented in other species. The architecture of MIR loci generating highly expressed 24-/21-nt sRNAs is featured by long terminal repeat—retrotransposons (LTR-RTs) in families of Ty3/Gypsy and Ty1/Copia elements. This implies that the production of sRNAs may be predominantly originated from TE fragments on chromosomes. Furthermore, a large proportion of highly expressed 24-nt sRNAs does not have predictable targets against unique genes in Picea, suggestive of their potential pathway in DNA methylation modifications on, for instance, TEs. Additionally, the classification of computationally predicted sRNAs suggests that 24-nt sRNA targets may bear particular functions in metabolic processes while 21-nt sRNAs target genes involved in many different biological processes. This study, therefore, directs our attention to a possible extrapolation that lacking of 24-nt sRNAs at the late conifer seed developmental phase may result in less constraints in TE activities, thus contributing to the massive expansion of genome size.

Abstract B18: Strand-specific in vivo screen of cancer-associated miRNAs unveils key drivers and oncogenic targets

Abstract MiRNAs are evolutionarily conserved small non-coding RNAs that orchestrate a wide spectrum of physiological and pathological processes in stem cells (SCs) and human cancers. Intimately wired into oncogenic and tumor suppressive networks, miRs are valuable as not only diagnostic biomarkers but also therapeutic targets. Squamous cell carcinomas (SCCs) are among the most life-threatening human cancers world-wide. Using skin as paradigm, we describe the first panoramic view of miRs during the development, homeostasis and malignant transformation of skin epithelium, whose SCs are the target of most common SCCs. Focusing on abundant and enriched miRs in SCC-SCs versus normal skin SCs, we devised lentiviral miR expression platform and conducted pooled in vivo functional screens for oncomiRs in mice. Deep sequencing of genomic DNA from basal progenitors of tumors or unaffected skin epithelium revealed preferential enrichment of 29/169 miRs within the tumors. We followed up with the top hits and showed candidate miRs are indeed aberrantly induced in both mouse and human SCCs. After functionally documenting their tumor-promoting roles in mouse SCCs in vivo and human xenografts, we applied CRISPR technology to ablate the miR loci in cells derived from these SCCs, and then used lentiviral delivery methods to carry out specific miR rescues of the tumorigenic phenotype. To look for candidate targets of newly identified oncomiRs, we analyzed TCGA database that are inversely correlated with our candidate miR as well as genes that are de-repressed upon miR deletion. We discovered potent tumor suppressors whose knockdown rescued the deficit in tumor formation from oncomiR-deficient cells and whose overexpression reversed miR-driven tumorigenesis. Furthermore, we demonstrated CRISPR-mediated deletion of the endogenous miRNA binding site renders the target cells no longer sensitive to miRNA regulation. Finally, we pinpointed the cellular mechanism of how miR-target regulates SCC progression. Empowered by mouse genetics and high throughput approaches, our study has unveiled a rich set of putative miRs that drive skin SCC malignancy, and discovered their oncogenic targets. These findings shed new light on increasingly important oncomiR loci and its action on a devastating cancer. Citation Format: Yejing Ge, Liang Zhang, Maria Nikolova, Boris Reva, Elaine Fuchs. Strand-specific in vivo screen of cancer-associated miRNAs unveils key drivers and oncogenic targets. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B18.

Abstract PR01: Strand-specific in vivo screen of cancer-associated miRNAs unveils key drivers and oncogenic targets

Abstract MiRNAs are evolutionarily conserved small non-coding RNAs that orchestrate a wide spectrum of physiological and pathological processes in stem cells (SCs) and human cancers. Intimately wired into oncogenic and tumor suppressive networks, miRs are valuable as not only diagnostic biomarkers but also therapeutic targets. Squamous cell carcinomas (SCCs) are among the most life-threatening human cancers world-wide. Using skin as paradigm, we describe the first panoramic view of miRs during the development, homeostasis and malignant transformation of skin epithelium, whose SCs are the target of most common SCCs. Focusing on abundant and enriched miRs in SCC-SCs versus normal skin SCs, we devised lentiviral miR expression platform and conducted pooled in vivo functional screens for oncomiRs in mice. Deep sequencing of genomic DNA from basal progenitors of tumors or unaffected skin epithelium revealed preferential enrichment of 29/169 miRs within the tumors. We followed up with the top hits and showed candidate miRs are indeed aberrantly induced in both mouse and human SCCs. After functionally documenting their tumor-promoting roles in mouse SCCs in vivo and human xenografts, we applied CRISPR technology to ablate the miR loci in cells derived from these SCCs, and then used lentiviral delivery methods to carry out specific miR rescues of the tumorigenic phenotype. To look for candidate targets of newly identified oncomiRs, we analyzed TCGA database that are inversely correlated with our candidate miR as well as genes that are de-repressed upon miR deletion. We discovered potent tumor suppressors whose knockdown rescued the deficit in tumor formation from oncomiR-deficient cells and whose overexpression reversed miR-driven tumorigenesis. Furthermore, we demonstrated CRISPR-mediated deletion of the endogenous miRNA binding site renders the target cells no longer sensitive to miRNA regulation. Finally, we pinpointed the cellular mechanism of how miR-target regulates SCC progression. Empowered by mouse genetics and high throughput approaches, our study has unveiled a rich set of putative miRs that drive skin SCC malignancy, and discovered their oncogenic targets. These findings shed new light on increasingly important oncomiR loci and its action on a devastating cancer. This abstract is also presented as Poster A02. Citation Format: Yejing Ge, Liang Zhang, Maria Nikolova, Boris Reva, Elaine Fuchs. Strand-specific in vivo screen of cancer-associated miRNAs unveils key drivers and oncogenic targets. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR01.

Genome-wide discovery and validation of Eucalyptus small RNAs reveals variable patterns of conservation and diversity across species of Myrtaceae.

BackgroundMicro RNAs are a class of small non coding RNAs of 20–24 nucleotides transcribed as single stranded precursors from MIR gene loci. Initially described as post-transcriptional regulators involved in development, two decades ago, miRNAs have been proven to regulate a wide range of processes in plants such as germination, morphology and responses to biotic and abiotic stress. Despite wide conservation in plants, a number of miRNAs are lineage specific. We describe the first genome wide survey of Eucalyptus miRNAs based on high throughput sequencing.ResultsIn addition to discovering small RNA sequences, MIR loci were mapped onto the reference genome and interspecific variability investigated. Sequencing was carried out for the two most world widely planted species, E. grandis and E. globulus. To maximize discovery, E. grandis samples were from BRASUZ1, the same tree whose genome provided the reference sequence. Interspecific analysis reinforces the variability in small RNA repertoire even between closely related species. Characterization of Eucalyptus small RNA sequences showed 95 orthologous to conserved miRNAs and 193 novel miRNAs. In silico target prediction confirmed 163 novel miRNAs and degradome sequencing experimentally confirmed several hundred targets. Experimental evidence based on the exclusive expression of a set of small RNAs across 16 species within Myrtaceae further highlighted variable patterns of conservation and diversity of these regulatory elements.ConclusionsThe description of miRNAs in Eucalyptus contributes to scientific knowledge of this vast genre, which is the most widely planted hardwood crop in the tropical and subtropical world, adding another important element to the annotation of Eucalyptus grandis reference genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2322-6) contains supplementary material, which is available to authorized users.

Continuing analysis of microRNA origins

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).

Identification and analysis of the proximal promoters of microRNA genes in Arabidopsis

Endogenous microRNAs (miRNAs) modulate gene expression at the post-transcription level. In plants, a vast majority of MIR genes are thought to be transcribed by RNA Polymerase II (Pol II). However, promoter organization is currently unknown for most plant MIR genes. This deficiency prevents a comprehensive understanding of miRNA-mediated gene networks. In this study, we performed Pol II chromatin immunoprecipitation (ChIP) analysis in Arabidopsis using a genome tiling microarray. Distinct Pol II binding was found at most MIR loci, which allowed prediction of the transcription start sites (TSSs) for 167 MIR genes in Arabidopsis that was validated by average free energy profiling. By employing 99 position weight matrices (PWM), we systematically scanned the regulatory regions upstream of the TSSs. We discovered eleven and ten cis-elements that are statistically over- and under-represented in MIR promoters, respectively. Thus, analysis of Pol II binding provides a new perspective for studying miRNAs in plants.

Dynamic expression of small RNA populations in larch (Larix leptolepis)

Small RNAs (sRNAs) are emerging as essential regulators of biological processes. However, several studies have reported that gymnosperms do not express appreciable amounts of 24-nt sRNAs, and conifers in particular may have a unique sRNA-silencing signature. Here, we compared the sRNA transcriptomes of Japanese larch somatic embryos (SE) and seedlings. SE sRNAs exhibited a length bias toward 24nt, while seedlings showed a bias toward a 21-nt length. We also confirmed that larch is capable of producing 24-nt sRNAs based on a polyacrylamide gel analysis. The sRNA expression patterns varied according to developmental stage, which might be associated with Dicer-like 3 and RNA-dependent RNA polymerase2 (RDR2) levels. Our data suggest that many MIR loci that produce canonical microRNAs (miRNAs, 20-22nt) and long sRNAs (23-26nt) have dual functions; the latter were preferentially produced in SE compared to seedlings. However, the ratio of miRNAs to total sRNAs in seedlings was higher than in SE, and most miRNAs were upregulated in seedlings. Trans-acting small interfering RNAs (ta-siRNAs) generated from TAS3 triggered by miR390 were identified, and levels of the three detected ta-siRNAs peaked in mature embryos, which was not consistent with the lowest RDR6 level. These findings indicate that larch, and possibly other gymnosperms, shares a common sRNA pathway with other land plants, and that the sRNA distribution pattern varies according to developmental stage, which may be attributable to the expression of sRNA pathway genes.

OrbId

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.

Comprehensive analysis of microRNA genomic loci identifies pervasive repetitive-element origins

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.

MiRTRAP, a computational method for the systematic identification of miRNAs from high throughput sequencing data

A novel method for prediction of miRs from deep sequencing data. Its utility is demonstrated when applied to Ciona data.

MicroRNA and cancer – focus on apoptosis

MicroRNAs (miRs) are small non-coding RNAs regulating gene expression at the post-transcriptional and/or translational levels. miRs play important roles in diverse biological processes, including development, cell differentiation, proliferation and apoptosis. Recent evidence has shown that miR loci frequently map to cancer-associated genomic regions and deregulated miR expression profiles are associated with many cancer types, implicating miRs in crucial processes that lead to tumourigenesis. Here, we review the current findings about miRs and tumourigenesis, focusing on their involvement in the apoptosis pathway. A significant observation is that greater than one-quarter of all known human miRs were reported to be deregulated in at least one cancer type. The expression of a subset of miRs (e.g. miR-21 and miR-155) was found to be consistently up-regulated, whereas another subset of miRs (e.g.miR-143 and miR-145) was consistently down-regulated across different cancer types suggesting their involvement in regulating common cellular processes whose deregulation may lead to tumourigenesis. Several miRs were implicated to play roles in cell proliferation and apoptosis. Some miRs, such as miR-29b and miR-15–16, influence only the apoptotic pathway, whereas others including let-7/miR-98 and miR-17–92 may play roles in both the apoptotic and cell-proliferation pathways. In conclusion, although our current understanding of the functions of miRs is still fragmentary, taken together, this review highlights the complex and intricate roles that miRs play in the regulation of cellular processes. Perturbation of the expression of miRs may thus lead to tumourigenesis.