Common micro RNAs (miRNAs) target complement factor H (CFH) regulation in Alzheimer’s disease (AD) and in age-related macular degeneration (AMD)

Alzheimer’s disease (AD) and age-related macular degeneration (AMD) are complex and progressive inflammatory degenerations of the human neocortex and retina. Recent molecular, genetic and epigenetic evidence indicate that at least 4 micro RNAs (miRNAs) - including the NF-кB-regulated miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 - are progressively up-regulated in both AD and AMD. This quartet of up-regulated miRNAs in turn down-regulate a small brain- and retinal-cell-relevant family of target mRNAs, including that encoding complement factor H (CFH), a major negative regulator of the innate immune and inflammatory response. Together miRNA-146a and miRNA-155 recognize an overlapping miRNA regulatory control (MiRC) region in the CFH 3’-untranslated region (3’- UTR; 5’-TTTAGTATTAA-3’) to which either of these miRNAs may interact. Progressive, pathogenic increases in specific miRNA binding to the entire 232 nucleotide CFH 3’-UTR appears to be a major regulator of CFH expression down-regulation, and the inflammatory pathology that characterizes both AMD and AD. The data presented in this report provides evidence that up-regulation of brain- and retinal- abundant miRNAs, including miRNA-9, miRNA-125b, miRNA-146a and miRNA-155, are common to the pathogenetic mechanism of CFH deficiency that drives inflammatory neurodegeneration, and for the first time indicates multiple, independent miRNA-mediated regulation of the CFH mRNA 3’-UTR.     

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the “RIP-Chip” assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.     

miRNA与其靶mRNA的相互作用：绑定位点的质量与数量特征的整合计算分析

miRNAs通过完全或不完全的碱基互补绑定到信使RNA(mRNA)上,通过抑制翻译或者直接导致mRNA降解的方式来调节靶基因的表达．为了研究miRNAs在转录水平上面的调控作用,两种人类基因组中组织特异的miRNAs(miR-1和miR-124)被转染到HeLa细胞中,微阵列(microarray)分析转染前后细胞中各基因mRNA表达水平变化情况的结果表明：动物基因组中靶基因与miRNAs不完全的碱基互补也会导致mRNA的直接降解．通过分析实验得到的mRNA表达水平变化数据,发现这相同miRNA的不同靶基因mRNA表达水平的下调倍数有着明显的差别,推测这些靶基因mRNA序列本身存在某些影响其受调节程度的因素．为此,提取和分析这些靶基因mRNA的序列特征,通过对这些序列特征与mRNA表达水平下调数据进行统计相关分析,最终发现,miRNA靶基因受调节的程度与以下几个因素相关联：mRNA序列中miRNA靶位点的个数,靶位点与miRNA序列碱基互补的程度,以及绑定后形成二级结构的稳定程度(即最低自由能的大小)．在此基础上,初步建立起一个多因子作用下的miRNA 靶基因mRNA表达水平下调程度模型,分析表明：该模型在一定程度上可以反映了部分序列特征对于miRNA靶基因mRNA表达水平下调程度的影响．     

Large-Scale Screens of miRNA-mRNA Interactions Unveiled That the 3′UTR of a Gene Is Targeted by Multiple miRNAs

Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3′ untranslated regions [3′UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3′UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3′UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3′ end targeted regions, as well as the 5′ end (seed region) binding sites.     

Uncoupling of lin-14 mRNA and protein repression by nutrient deprivation in Caenorhabditis elegans

In animals, microRNAs (miRNAs), typically, pair to sites of partial complementarity in the 3′-untranslated regions (3′UTRs) of target genes. Regulation by miRNAs often results in down-regulation of target mRNA and protein expression by mechanisms that are yet to be fully elucidated. Additionally, changes in environmental conditions have been shown to influence miRNA function in some cell culture systems. Here, we report the effect of nutrient deprivation on regulation of an endogenous miRNA target in developing worms. In Caenorhabditis elegans, the lin-4 miRNA recognizes multiple sites in the lin-14 3′UTR and directs mRNA degradation and translational repression, but it is unclear how these processes are coupled. In this study, we demonstrate that nutrient deprivation results in loss of lin-14 mRNA, but not protein, repression. In worms removed from feeding conditions, lin-14 mRNA reaccumulates despite the continued expression of lin-4 miRNA. The relative increase in lin-14 mRNA levels during nutrient deprivation is less pronounced in genetic mutants lacking lin-4 miRNA or the lin-14 3′UTR target sites. In conclusion, regulation of lin-14 at the mRNA and protein levels can be uncoupled by changes in culture conditions, indicating that miRNA function can be modulated by environment in multicellular organisms. The awareness that endogenous miRNA pathways can be sensitive to environment is an important consideration for elucidating the mechanism used by miRNAs to regulate target mRNA and protein expression.     

Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4)

Recent small RNA sequencing data has uncovered 3′ end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3′ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3′ mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3′ mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3′ miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.     

A meta-analysis revealed insights into the sources,conservation and impact of microRNA 5′-isoforms in four model species

MicroRNA (miRNA) 5′-isoforms, or 5′-isomiRs, are small-RNA species that originate from the same genomic loci as the major miRNAs with their 5′ ends shifted from the 5′ ends of the miRNAs by a few nucleotides. Although 5′-isomiRs have been reported, their origins, properties and potential functions remain to be examined. We systematically studied 5′-isomiRs in human, mouse, fruitfly and worm by analysing a large collection of small non-coding RNA and mRNA profiling data. The results revealed a broad existence of 5′-isomiRs in the four species, many of which were conserved and could arise from genomic loci of canonical and non-canonical miRNAs. The well-conserved 5′-isomiRs have several features, including a preference of the 3p over the 5p arms of hairpins of conserved mammalian miRNAs, altered 5′-isomiRs across species and across tissues, and association with structural variations of miRNA hairpins. Importantly, 5′-isomiRs and their major miRNAs may have different mRNA targets and thus potentially play distinct roles of gene regulation, as shown by an integrative analysis combining miRNA and mRNA profiling data from psoriatic and normal human skin and from murine miRNA knockout assays. Indeed, 18 5′-isomiRs had aberrant expression in psoriatic human skin, suggesting their potential function in psoriasis pathogenesis. The results of the current study deepened our understanding of the diversity and conservation of miRNAs, their plasticity in gene regulation and potential broad function in complex diseases.     

The Impact of 3′UTR Variants on Differential Expression of Candidate Cancer Susceptibility Genes

Variants in regulatory regions are predicted to play an important role in disease susceptibility of common diseases. Polymorphisms mapping to microRNA (miRNA) binding sites have been shown to disrupt the ability of miRNAs to target genes resulting in differential mRNA and protein expression. Skin tumor susceptibility 5 (Skts5) was identified as a locus conferring susceptibility to chemically-induced skin cancer in NIH/Ola by SPRET/Outbred F1 backcrosses. To determine if polymorphisms between the strains which mapped to putative miRNA binding sites in the 3′ untranslated region (3′UTR) of genes at Skts5 influenced expression, we conducted a systematic evaluation of 3′UTRs of candidate genes across this locus. Nine genes had polymorphisms in their 3′UTRs which fit the linkage data and eight of these contained polymorphisms suspected to interfere with or introduce miRNA binding. 3′UTRs of six genes, Bcap29, Dgkb, Hbp1, Pik3cg, Twistnb, and Tspan13 differentially affected luciferase expression, but did not appear to be differentially regulated by the evaluated miRNAs predicted to bind to only one of the two isoforms. 3′UTRs from four additional genes chosen from the locus that fit less stringent criteria were evaluated. Ifrd1 and Etv1 showed differences and contained polymorphisms predicted to disrupt or create miRNA binding sites but showed no difference in regulation by the miRNAs tested. In summary, multiple 3′UTRs with putative functional variants between susceptible and resistant strains of mice influenced differential expression independent of predicted miRNA binding.