The pacemaker properties of the suprachiasmatic nucleus (SCN) circadian clock are shaped by mechanisms that influence the expression and behavior of clock proteins. Here, we reveal that G-protein-coupled receptor kinase 2 (GRK2) modulates the period, amplitude, and entrainment characteristics of the SCN. Grk2-deficient mice show phase-dependent alterations in light-induced entrainment, slower recovery from jetlag, and longer behavioral rhythms. Grk2 ablation perturbs intrinsic rhythmic properties of the SCN, increasing amplitude and decreasing period. At the cellular level, GRK2 suppresses the transcription of the mPeriod1 gene and the trafficking of PERIOD1 and PERIOD2 proteins to the nucleus. Moreover, GRK2 can physically interact with PERIOD1/2 and promote PERIOD2 phosphorylation at Ser545, effects that may underlie its ability to regulate PERIOD1/2 trafficking. Together, our findings identify GRK2 as an important modulator of circadian clock speed, amplitude, and entrainment by controlling PERIOD at the transcriptional and post-translational levels.

The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of the hippocampus.

Caveolin-1 (cav-1), an integral protein of the membrane microdomains caveolae, is required for synthesis of matrix proteins by glomerular mesangial cells (MC). Previously, we demonstrated that the antifibrotic protein follistatin (FST) is transcriptionally upregulated in cav-1 knockout MC and that its administration is protective against renal fibrosis. Here, we screened cav-1 wild-type and knockout MC for FST-targeting microRNAs in order to identity novel antifibrotic therapeutic targets. We identified that miR299a-5p was significantly suppressed in cav-1 knockout MC, and this was associated with stabilization of the FST 3'UTR. Overexpression and inhibition studies confirmed the role of miR299a-5p in regulating FST expression. Furthermore, the profibrotic cytokine TGFβ1 was found to stimulate the expression of miR299a-5p and, in turn, downregulate FST. Through inhibition of FST, miR299a-5p overexpression augmented, while miR299a-5p inhibition diminished TGFβ1 profibrotic responses, whereas miR299a-5p overexpression re-enabled cav-1 knockout MC to respond to TGFβ1. In vivo, miR299a-5p was upregulated in the kidneys of mice with chronic kidney disease (CKD). miR299a-5p inhibition protected these mice against renal fibrosis and CKD severity. Our data demonstrate that miR299a-5p is an important post-transcriptional regulator of FST, with its upregulation an important pathogenic contributor to renal fibrosis. Thus, miR299a-5p inhibition offers a potential novel therapeutic approach for CKD.

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the 'A10234287911' allele may promote better processing vis-à-vis the structure conferred by 'G10234287911' allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry. Of the 2112 proteins that were accurately quantified, 20% (421 proteins) displayed a time-of-day-dependent expression profile. Within this time-of-day proteome, 11% (48 proteins) were further defined as circadian based on a sinusoidal expression pattern with a ∼24 h period. Nine circadianly expressed proteins exhibited 24 h rhythms at the transcript level, with an average time lag that exceeded 8 h. A substantial proportion of the time-of-day proteome exhibited abrupt fluctuations at the anticipated light-to-dark and dark-to-light transitions, and was enriched for proteins involved in several key biological pathways, most notably, mitochondrial oxidative phosphorylation. Additionally, predicted targets of miR-133ab were enriched in specific hierarchical clusters and were inversely correlated with miR133ab expression in the SCN. These insights into the proteomic landscape of the SCN will facilitate a more integrative understanding of cellular control within the SCN clock.

Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate.

Sterol regulatory element binding protein (SREBP) is an important potential mediator of kidney fibrosis and is known to be upregulated in diabetic nephropathy. We evaluated the effectiveness of SREBP inhibition as treatment of diabetic nephropathy. Type 1 diabetes was induced in uninephrectomized male CD1 mice with streptozotocin. The mice were treated with the SREBP inhibitor fatostatin for 12 weeks. At the endpoint, kidney function and pathologic findings were assessed. Fatostatin inhibited the increase of both isoforms of SREBP (types 1 and 2) in diabetic kidneys. Treatment attenuated basement membrane thickening but did not improve hyperfiltration, albuminuria, or kidney fibrosis in diabetic mice. The treatment of nondiabetic mice with fatostatin led to hyperfiltration and increased the glomerular volume to levels seen in diabetic mice. This was associated with increased renal inflammation and a trend toward increased renal fibrosis. Both in vivo and in cultured renal proximal tubular epithelial cells, fatostatin increased the expression of the proinflammatory cytokine monocyte chemoattractant protein-1. Thus, SREBP inhibition with fatostatin not only is ineffective in preventing diabetic nephropathy but also leads to kidney injury in nondiabetic mice. Further research on the efficacy of other SREBP inhibitors and the specific roles of SREBP-1 and SREBP-2 in the treatment and pathogenesis of diabetic nephropathy is needed.

We previously showed that caveolin-1 (cav-1), an integral membrane protein, is required for the synthesis of matrix proteins by glomerular mesangial cells (MC). In a previous study to understand how cav-1 is involved in regulating matrix production, we had identified significant upregulation of the antifibrotic protein follistatin in cav-1 knockout MC. Follistatin inhibits the profibrotic effects of several members of the transforming growth factor beta superfamily, in particular the activins. Here, we characterize the molecular mechanism through which cav-1 regulates the expression of follistatin.

Naturally evolved saline tolerant rice landraces found along the coastline of India are a valuable genomic resource to explore the complex, polygenic nature of salinity tolerance. In the present study, a set of 28 genome wide SSR markers, 11 salt responsive genic SSR markers and 8 Saltol QTL linked SSR markers were used to estimate genetic relatedness and population structure within a collection of 47 rice landraces (including a tolerant and 2 sensitive checks) originating from geographically divergent coastal regions of India. All three marker types identified substantial genetic variation among the landraces, as evident from their higher PIC values (0.53 for genomic SSRs, 0.43 for Genic SSRs and 0.59 for Saltol SSRs). The markers RM431, RM484 (Genomic SSRs), OsCAX (D), OsCAX (T) (Genic SSRs) and RM562 (Saltol SSR) were identified as good candidates to be used in breeding programs for improving salinity tolerance in rice. STRUCTURE analysis divided the landraces into five distinct populations, with classification correlating with their geographical locations. Principal coordinate and hierarchical cluster analyses (UPGMA and neighbor joining) are in close agreement with STRUCTURE results. AMOVA analysis indicated a higher magnitude of genetic differentiation within individuals of groups (58%), than among groups (42%). We also report the development and validation of a new Cleavage Amplified Polymorphic Sequence (CAPS) marker (OsHKT1;5V395) that targets a codon in the sodium transporter gene OsHKT1;5 (Saltol/SKC1 locus) that is associated with sodium transport rates in the above rice landraces. The CAPS marker was found to be present in all landraces except in IR29, Kamini, Gheus, Matla 1 and Matla 2. Significant molecular genetic diversity established among the analyzed salt tolerant rice landraces will aid in future association mapping; the CAPS marker, OsHKT1;5V395 can be used to map rice landraces for the presence of the SNP (Single Nucleotide Polymorphism) associated with increased sodium transport rates and concomitant salinity tolerance in rice.

Glomerular matrix protein accumulation, mediated largely by mesangial cells, is central to the pathogenesis of diabetic kidney disease. Our previous studies showed that the membrane microdomains caveolae and their marker protein caveolin-1 regulate matrix protein synthesis in mesangial cells in response to diabetogenic stimuli, and that caveolin-1 knockout mice are protected against diabetic kidney disease. In a screen to identify the molecular mechanism underlying this protection, we also established that secreted antifibrotic glycoprotein follistatin is significantly upregulated by caveolin-1 deletion. Follistatin potently neutralizes activins, members of the transforming growth factor-β superfamily. A role for activins in diabetic kidney disease has not yet been established. Therefore, in vitro, we confirmed the regulation of follistatin by caveolin-1 in primary mesangial cells and showed that follistatin controls both basal and glucose-induced matrix production through activin inhibition. In vivo, we found activin A upregulation by immunohistochemistry in both mouse and human diabetic kidney disease. Importantly, administration of follistatin to type 1 diabetic Akita mice attenuated early diabetic kidney disease, characterized by albuminuria, hyperfiltration, basement membrane thickening, loss of endothelial glycocalyx and podocyte nephrin, and glomerular matrix accumulation. Thus, activin A is an important mediator of high glucose-induced profibrotic responses in mesangial cells, and follistatin may be a potential novel therapy for the prevention of diabetic kidney disease.