Expression and Rhythmic Modulation of Circulating MicroRNAs Targeting the Clock Gene Bmal1 in Mice

MicroRNAs (miRNAs) interact with 3′ untranslated region (UTR) elements of target genes to regulate mRNA stability or translation and thus play a role in regulating many different biological processes, including circadian rhythms. However, specific miRNAs mediating the regulation of essential clock genes remain largely unknown. Because vesicles containing membrane-bound miRNAs are present in the circulatory system, we examined miRNAs predicted to target the clock gene, Bmal1, for evidence of rhythmic fluctuations in circulating levels and modulatory effects on the 3′ UTR activity of Bmal1. A number of miRNAs with Bmal1 as a predicted target were expressed in the serum of mice exposed to LD 12∶12 and of these miRNAs, miR-152 and miR-494 but not miR-142-3p were marked by diurnal oscillations with bimodal peaks in expression occurring near the middle of the day and 8 or 12 hr later during the night. Co-transfection of pre-miR over-expression constructs for miR-494 and miR-142-3p in HEK293 cells had significant effects in repressing luciferase-reported Bmal1 3′ UTR activity by as much as 60%, suggesting that these miRNAs may function as post-transcriptional modulators of Bmal1. In conjunction with previous studies implicating miRNAs as extracellular regulatory signals, our results suggest that circulating miRNAs may play a role in the regulation of the molecular clockworks in peripheral circadian oscillators

Role of miR-142-3p in the post-transcriptional regulation of the clock gene Bmal1 in the mouse SCN.

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional modulators by regulating stability or translation of target mRNAs. Recent studies have implicated miRNAs in the regulation of mammalian circadian rhythms. To explore the role of miRNAs in the post-transcriptional modulation of core clock genes in the master circadian pacemaker, we examined miR-142-3p for evidence of circadian expression in the suprachiasmatic nuclei (SCN), regulation of its putative clock gene target Bmal1 via specific binding sites in the 3' UTR and overexpression-induced changes in the circadian rhythm of BMAL1 protein levels in SCN cells. In mice exposed to constant darkness (DD), miR-142-3p levels in the SCN were characterized by circadian rhythmicity with peak expression during early subjective day at CT 3. Mutagenesis studies indicate that two independent miRNA recognition elements located at nucleotides 1-7 and 335-357 contribute equally to miR-142-3p-induced repression of luciferase-reported Bmal1 3' UTR activity. Importantly, overexpression of miR-142-3p in immortalized SCN cells abolished circadian variation in endogenous BMAL1 protein levels in vitro. Collectively, our results suggest that miR-142-3p may play a role in the post-transcriptional modulation of Bmal1 and its oscillatory regulation in molecular feedback loops mediating SCN circadian function

Effects of binding site mutagenesis on miR-142-3p-induced repression of <i>Bmal1</i> 3′ UTR activity.

<p>(A) Diagrammatic representation of WT and mutagenized <i>Bmal1</i> 3′ UTR constructs with independent site-directed deletions of predicted miR-142-3p binding sites. (B) Normalized bioluminescence from HEK293 cells co-transfected with pEZX-MR04 miR-142 expression vector and with either the target control, full-length <i>Bmal1</i> (WT), <i>Bmal1 c.1_7del, Bmal1 c.335_357del</i> or <i>Bmal1 c.1_7del</i>+<i>c.335_357del</i> miRNA 3′ UTR target clones. (C) Normalized bioluminescence from HEK293 cells co-transfected with pEZX-MR04 miR-494 expression vector with either the target control, full-length <i>Bmal1</i> (WT), <i>Bmal1 c.1_7del, Bmal1 c.335_357del</i> or <i>Bmal1 c.1_7del</i>+<i>c.335_357del</i> miRNA 3′ UTR target clones. Bars represent mean (±SEM) determinations of luciferase bioluminescence for each treatment group (n = 4). The plotted values correspond to the ratios of firefly luciferase signal normalized to <i>Renilla</i> luciferase activity in the same sample and are represented as a percentage of the average signal for control vector transfectants. Asterisks denote comparisons in which normalized bioluminescence for a given treatment group was significantly reduced (<i>p</i><0.05) relative to that observed in control vector transfectants.</p

Effects of miR-142-3p overexpression on the circadian rhythm of BMAL1 protein content in immortalized SCN cells <i>in vitro</i>.

<p>Representative Western blot results and densitometric determinations of BMAL1 protein levels at 4-hour intervals in cultures (n = 4–5) of <i>mPer2^Luc</i> SCN cells transfected with pEZX-MR04 control miRNA expression vector (<b>CONT</b>) or pEZX-MR04 miR-142 expression vector (<b>miR-142</b>). The plotted values represent the relative optical density (mean ± SEM) and correspond to the ratios of BMAL1/β-actin immunoreactive signal in each sample. The asterisk indicates that the peak in BMAL1 protein levels at 20 hr was significantly greater (<i>p</i><0.05) than that observed during succeeding minima.</p

Temporal patterns of miR-142-3p and <i>Bmal1</i> expression in the mouse SCN.

<p>Symbols denote real-time PCR determinations (mean ± SEM) of miR-142-3p (red) and <i>Bmal1</i> mRNA (black) levels at 4-hour intervals in the SCN (n = 4-5) of mice exposed to constant darkness (DD). The plotted values correspond to the ratios of miR-142-3p signal normalized to U6 snRNA levels and of <i>Bmal1</i>/<i>Ppia</i> mRNA signal in which the maximal value for each gene was set at 100%. Asterisks indicate time points during which peak levels were significantly greater (<i>p</i><0.05) than those observed during preceding or succeeding minima.</p

Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (−)-Eurotiumin A

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to &gt;99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield

Structural and Data Science-Driven Analysis to Assess Substrate Specificity of Diketopiperazine Reverse Prenyltransferase NotF: Cascade Biocatalytic Synthesis of (–)-Eurotiumin A

Prenyltransfer is an early-stage carbon–hydrogen bond (C–H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Towards the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). In the biosynthesis of cytotoxic notoamide metabolites, PT NotF is responsible for catalyzing the first tailoring event of C2 reverse prenyltransfer of brevianamide F (cyclo(L-Trp-L-Pro)). Obtaining a high-resolution crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent exposed substrate binding site, intimating NotF may possess significant substrate promiscuity. To assess the full potential of NotF’s broad substrate selectivity, we synthesized a panel of 30 tryptophanyl DKPs with a suite of sterically and electronically differentiated amino acids, which were selectively prenylated by NotF in often synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library enabled the development of a descriptive statistical model that provided insight into the origins of NotF’s substrate promiscuity. Through this unique approach for understanding enzyme scope, we identified key substrate descriptors such as electrophilicity, size, and flexibility, that govern enzymatic turnover by NotF. Additionally, we demonstrated the ability to couple NotF-catalyzed prenyltransfer with oxidative cyclization using recently characterized flavin monooxygenase, BvnB, from the brevianamide biosynthetic pathway. This one-pot, in vitro biocatalytic cascade proceeds with exceptional substrate recognition, and enabled the first chemoenzymatic synthesis of the marine fungal natural product, (–)-eurotiumin A, in three steps and 60% overall yield

A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TE_S148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TE_S148C and TE_WT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TE_WT) to a lower energy concerted acyl substitution (TE_S148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones

A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TE_S148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TE_S148C and TE_WT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TE_WT) to a lower energy concerted acyl substitution (TE_S148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones