An SREBP-Responsive microRNA Operon Contributes to a Regulatory Loop for Intracellular Lipid Homeostasis

SummarySterol regulatory element-binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic microRNA (miRNA) locus that is activated directly by SREBP-2. Two of the encoded miRNAs, miR-182 and miR-96, negatively regulate the expression of Fbxw7 and Insig-2, respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miRNA pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a mechanism for the regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRNAs that are expressed from the same SREBP-2-regulated miRNA locus, and each targets a different protein of the multistep pathway that regulates SREBP function. These studies reveal an miRNA “operon” analogous to the classic model for genetic control in bacterial regulatory systems

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies

MicroRNA-122 Modulates the Rhythmic Expression Profile of the Circadian Deadenylase Nocturnin in Mouse Liver

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3′-untranslated region (3′-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3′-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control

Roles of Cabin one and Left one in T-cell development

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.Includes bibliographical references.Cabinl binds calcineurin and myocyte enhancer factor 2 (MEF2) through the C-terminal region. In cell lines, these interactions inhibit calcineurin activity following TCR signaling and transcriptional activation of Nur77 by MEF2. The role of these interactions in vivo was investigated using a mutant mouse strain that expresses a truncated Cabin 1 lacking the C-terminal calcineurin and MEF2 binding domains. Although mutant mice exhibited normal lymphocyte development and thymocyte apoptosis, they had elevated levels of serum IgG 1, IgG2b and IgE and produced more IgG1 and IgG2b in response to a T-dependent antigen. The increased antibody production is apparently not due to changes in immunoglobulin class switching as B cells from mutant mice switched to IgG 1 at the same frequency as wildtype B cells in vitro in the presence of IL-4. However, in response to anti-CD3 stimulation, mutant T cells expressed significantly higher levels of IL-2, IL-4, IL-9, IL-13, and IFN-y. Thus, the calcineurin and MEF2 binding domain of Cabin 1 is dispensable for thymocyte development and apoptosis, but is required for proper regulation of T cell cytokine expression and Th2 antibody responses. In our analysis of gene expression in memory CD8+ T cells, we identified a member of the membrane spanning 4A gene family, MS4a4b, which we named Leftl. It was also found to be expressed in Thl, but not Th2 cells. To examine the role of Leftl in T cell development, we created transgenic mice which express Leftl in T cells. These mice revealed a role for Leftl at multiple stages of T cell development.(cont.) Ectopic expression of Leftl in CD4+CD8+ thymocytes promoted CD8+ lineage commitment, although CD4+ thymocyte development was unaffected. Although mature transgenic T cells were impaired in their response to certain stimuli in vitro, there was a dramatic increase in the proportion of memory-phenotype T cells in vivo. Finally, Th2 cytokine and GATA-3 expression was impaired in Leftl transgenic Th2 cells. Transgenic T cells activated under Th2 polarizing conditions maintained some characteristics of Thl cells, such as the expression of T-bet and IL-12 receptor. Downregulation of Leftl expression is an essential step in commitment to the Th2 lineage.by Christine C. Esau.Ph.D

Effect of miR-122 on <i>Nocturnin</i> reporter expression is specific.

<p><b>A</b>. Relative luciferase activities (means ± S.E. Two independent experiments in triplicate) when reporter genes were co-transfected with either miR-122, miR-125a, or miR-125b in NIH3T3 cells. Asterisks represent p<0.005 miR-122 versus no miR-122, miR-125a, or miR-125b. <b>B</b>. Northern blot analysis of miRNAs. Same amount of plasmid DNA (1 µg) to express miR-122, -125a, and -125b was transfected into HEK293 cells.</p

The <i>Nocturnin</i> 3′UTR possesses one putative miR-122 recognition site.

<p><b>A</b>. The sequence of WT <i>Nocturnin</i> 3′-UTR (top) and miR-122 (bottom) around the putative miR-122 recognition site. Black and gray lines represent the perfect match and G-U wobbles, respectively. <b>B</b>. miR-122 recognition sequences of <i>Nocturnin</i> gene in Homo sapiens (NM_012118; nt1930–1953), Mus musculus (NM_009834, nt2100–2121), Rattus norvegicus (NM_138526, nt1680–1702), and Bos taurus (NM_001082454, nt1693–1715). Red characters represent the seed sequence for miR-122 recognition.</p

Deadenylase activity of <i>Nocturnin</i> was not involved in miR-122-mediated self-regulation.

<p><b>A</b>. Relative luciferase activities (means ± S.E. Three independent experiments in duplicate) with miR-122 overexpression in MEFs (White bars; +/+, Gray bars; +/−, Black bars; −/−). Luciferase activities were normalized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011264#pone-0011264-g002" target="_blank">Figure 2</a>. Asterisks represent p<0.005 versus no miR-122. <b>B</b>. miR-122 expression was measured from <i>Noc</i>^+/+ and <i>Noc</i>^−/− livers. RNA samples are pools of equal amounts of RNA from each circadian time point (ZT 0, 4, 8, 12, 16, and 20, one mouse per time point).</p

Nocturnin is up-regulated by miR-122 knock-down <i>in vivo</i>.

<p><b>A</b>. <i>Nocturnin</i> mRNA expression was measured by qRT-PCR from livers collected at the times indicated from mice that were previously treated with miR-122 ASO or PBS (mean ± S.E.). Each time point represents an average from three mice. p<0.05 by two-way ANOVA (PBS vs. miR-122 ASO). <b>B</b>. Nocturnin protein expression in PBS or miR-122 ASO treated mouse liver (mean ± S.E.) around the clock was measured on Western blots and then quantitated using Image J software. Each point represents average of three mice. Two-way ANOVA (PBS vs. miR-122 ASO) was not significant (p = 0.32). <b>C</b>. Representative Western blots of Nocturnin expression in PBS, miR-122 ASO, or miR-124 ASO treated mouse liver (two mice per group) at ZT0 and ZT12. An independent set of mice as that in panels A and B was used. <b>D</b>. Nocturnin protein expression in PBS, miR-122 ASO, or miR-124 ASO treated mouse liver (mean ± S.E.) at ZT0 and ZT12. Western blots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011264#pone-0011264-g006" target="_blank">Fig. 6C</a>) were quantitated using Image J software. Each point represents an average of two mice.</p