Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse.

The adaptive response to extreme endurance exercise might involve transcriptional and translational regulation by microRNAs (miRNAs). Therefore, the objective of the present study was to perform an integrated analysis of the blood transcriptome and miRNome (using microarrays) in the horse before and after a 160 km endurance competition. A total of 2,453 differentially expressed genes and 167 differentially expressed microRNAs were identified when comparing pre- and post-ride samples. We used a hypergeometric test and its generalization to gain a better understanding of the biological functions regulated by the differentially expressed microRNA. In particular, 44 differentially expressed microRNAs putatively regulated a total of 351 depleted differentially expressed genes involved variously in glucose metabolism, fatty acid oxidation, mitochondrion biogenesis, and immune response pathways. In an independent validation set of animals, graphical Gaussian models confirmed that miR-21-5p, miR-181b-5p and miR-505-5p are candidate regulatory molecules for the adaptation to endurance exercise in the horse. To the best of our knowledge, the present study is the first to provide a comprehensive, integrated overview of the microRNA-mRNA co-regulation networks that may have a key role in controlling post-transcriptomic regulation during endurance exercise.

Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications.

The cardiac voltage-gated Na(+) channel, Na(V)1.5, is responsible for the upstroke of the action potential in cardiomyocytes and for efficient propagation of the electrical impulse in the myocardium. Even subtle alterations of Na(V)1.5 function, as caused by mutations in its gene SCN5A, may lead to many different arrhythmic phenotypes in carrier patients. In addition, acquired malfunctions of Na(V)1.5 that are secondary to cardiac disorders such as heart failure and cardiomyopathies, may also play significant roles in arrhythmogenesis. While it is clear that the regulation of Na(V)1.5 protein expression and function tightly depends on genetic mechanisms, recent studies have demonstrated that Na(V)1.5 is the target of various post-translational modifications that are pivotal not only in physiological conditions, but also in disease. In this review, we examine the recent literature demonstrating glycosylation, phosphorylation by Protein Kinases A and C, Ca(2+)/Calmodulin-dependent protein Kinase II, Phosphatidylinositol 3-Kinase, Serum- and Glucocorticoid-inducible Kinases, Fyn and Adenosine Monophosphate-activated Protein Kinase, methylation, acetylation, redox modifications, and ubiquitylation of Na(V)1.5. Modern and sensitive mass spectrometry approaches, applied directly to channel proteins that were purified from native cardiac tissues, have enabled the determination of the precise location of post-translational modification sites, thus providing essential information for understanding the mechanistic details of these regulations. The current challenge is first, to understand the roles of these modifications on the expression and the function of Na(V)1.5, and second, to further identify other chemical modifications. It is postulated that the diversity of phenotypes observed with Na(V)1.5-dependent disorders may partially arise from the complex post-translational modifications of channel protein components.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

Regulation of androgen biosynthesis - A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model

Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.