Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in the heart and lungs, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in cotranscribed clusters and examples in which TDMD underlies "arm switching," a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8-null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8 , which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

MicroRNA turnover: a tale of tailing, trimming, and targets.

MicroRNAs (miRNAs) post-transcriptionally repress gene expression by guiding Argonaute (AGO) proteins to target mRNAs. While much is known about the regulation of miRNA biogenesis, miRNA degradation pathways are comparatively poorly understood. Although miRNAs generally exhibit slow turnover, they can be rapidly degraded through regulated mechanisms that act in a context- or sequence-specific manner. Recent work has revealed a particularly important role for specialized target interactions in controlling rates of miRNA degradation. Engagement of these targets is associated with the addition and removal of nucleotides from the 3' ends of miRNAs, a process known as tailing and trimming. Here we review these mechanisms of miRNA modification and turnover, highlighting the contexts in which they impact miRNA stability and discussing important questions that remain unanswered.

A long term study of the difference in efficacy and effect rate of various concentrations of retinol (1500-6600 IU) in middle aged women.

Retinol is widely used for topical application for antiaging. However, the efficacy and effect rate of different concentrations of retinol have been rarely analyzed. Therefore, in this study, the efficacy and rate of effect of retinol concentrations from 1500 to 6600 IU, on various skin parameters, have been compared. Seventy-two Korean women aged 40-59 years participated in this study. Retinol was used by them for 24 weeks; the effects were measured at 0, 2, 4, 8, 12, and 24 weeks. The measurement parameters for aging were crow's feet, forehead wrinkles, nasolabial fold, dermal density, and elasticity and that for skin color were skin brightness, yellowness, redness, and standard deviation of skin brightness. The texture of the skin was measured by measuring the skin roughness and pores, and the skin barrier function was evaluated through hydration, sebum, and desquamation. Low concentration retinol (1500-2500 IU) had a significantly higher effect in skin color, brightness, and elasticity and faster improvement rate in skin brightness and elasticity compared to that for high concentration (3300-6600 IU). High concentration of retinol had a significantly higher effect in wrinkles, dermal density and pores and faster improvement rate for wrinkles, skin texture, pores, and desquamation compared to that for low concentration. This study evaluated the changes caused by different concentration of retinol over a long period of time. The results of this study have great implications as the optimal concentration of retinol can be prescribed for an accurate period for the desired results without side effects.

A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming.

MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process called target-directed miRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. We identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adaptor ZSWIM8, that mediates TDMD. The ZSWIM8 CRL interacts with AGO proteins, promotes TDMD in a tailing and trimming-independent manner, and regulates miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.

Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine.

Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.

Suppression of Ribosomal Pausing by eIF5A Is Necessary to Maintain the Fidelity of Start Codon Selection.

Sequences within 5' UTRs dictate the site and efficiency of translation initiation. In this study, an unbiased screen designed to interrogate the 5' UTR-mediated regulation of the growth-promoting gene MYC unexpectedly revealed the ribosomal pause relief factor eIF5A as a regulator of translation initiation codon selection. Depletion of eIF5A enhances upstream translation within 5' UTRs across yeast and human transcriptomes, including on the MYC transcript, where this results in increased production of an N-terminally extended protein. Furthermore, ribosome profiling experiments established that the function of eIF5A as a suppressor of ribosomal pausing at sites of suboptimal peptide bond formation is conserved in human cells. We present evidence that proximal ribosomal pausing on a transcript triggers enhanced use of upstream suboptimal or non-canonical initiation codons. Thus, we propose that eIF5A functions not only to maintain efficient translation elongation in eukaryotic cells but also to maintain the fidelity of translation initiation.

Conservation of mRNA quality control factor Ski7 and its diversification through changes in alternative splicing and gene duplication.

Eukaryotes maintain fidelity of gene expression by preferential degradation of aberrant mRNAs that arise by errors in RNA processing reactions. In Saccharomyces cerevisiae, Ski7 plays an important role in this mRNA quality control by mediating mRNA degradation by the RNA exosome. Ski7 was initially thought to be restricted to Saccharomyces cerevisiae and close relatives because the SKI7 gene and its paralog HBS1 arose by whole genome duplication (WGD) in a recent ancestor. We have recently shown that the preduplication gene was alternatively spliced and that Ski7 function predates WGD. Here, we use transcriptome analysis of diverse eukaryotes to show that diverse eukaryotes use alternative splicing of SKI7/HBS1 to encode two proteins. Although alternative splicing affects the same intrinsically disordered region of the protein, the pattern of splice site usage varies. This alternative splicing event arose in an early eukaryote that is a common ancestor of plants, animals, and fungi. Remarkably, through changes in alternative splicing and gene duplication, the Ski7 protein has diversified such that different species express one of four distinct Ski7-like proteins. We also show experimentally that the Saccharomyces cerevisiae SKI7 gene has undergone multiple changes that are incompatible with the Hbs1 function and may also have undergone additional changes to optimize mRNA quality control. The combination of transcriptome analysis in diverse eukaryotes and genetic analysis in yeast clarifies the mechanism by which a Ski7-like protein is expressed across eukaryotes and provides a unique view of changes in alternative splicing patterns of one gene over long evolutionary time.

The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions.

The RNA exosome is a 3'-5' ribonuclease complex that is composed of nine core subunits and an essential catalytic subunit, Rrp44. Two distinct conformations of Rrp44 were revealed in previous structural studies, suggesting that Rrp44 may change its conformation to exert its function. In the channeling conformation, (Rrp44(ch)), RNA accesses the active site after traversing the central channel of the RNA exosome, whereas in the other conformation, (Rrp44(da)), RNA gains direct access to the active site. Here, we show that the Rrp44(da) exosome is important for nuclear function of the RNA exosome. Defects caused by disrupting the direct access conformation are distinct from those caused by channel-occluding mutations, indicating specific functions for each conformation. Our genetic analyses provide in vivo evidence that the RNA exosome employs a direct-access route to recruit specific substrates, indicating that the RNA exosome uses alternative conformations to act on different RNA substrates.

Phosphorylation regulates mycobacterial proteasome.

Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome ß-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

The Association between Symptoms of Autonomic Neuropathy and the Heart Rate Variability in Diabetics.

BACKGROUND: There are few tools to detect the diabetic autonomic neuropathy at an earlier stage. This study was conducted to investigate the association between symptoms of autonomic neuropathy and the heart rate variability (HRV) in diabetics. METHODS: Study subjects consisted of 50 diabetic patients and 30 outpatient hospital control patients at a university family medicine department. The patients completed a Korean version of composite autonomic symptom scale (COMPASS). Electrocardiography was recorded in the supine position, on standing, and during deep breathing, for 5 minutes each. HRV of frequency domain was calculated by power spectral analysis. RESULTS: The COMPASS score was higher in female diabetic patients compared with that in controls. Among 50 diabetic patients, the total COMPASS score correlated positively with normalized low frequency (LF) score (normalized units, n.u.) (r = 0.62, P < 0 .001) and low frequency/high frequency (LF/HF) (r = 0.77, P < 0.001), negatively with normalized HF score (n.u.) (r = -0.59, P < 0.001) and RMSSD (square root of the mean of the sum of the square of differences between adjacent NN interval; r = -0.33, P = 0.031). The decrease in LF (n.u) and the increase in HF (n.u) by deep breathing from the supine position were higher in diabetic patients compared with those in controls. The increase in LF (n.u) and the decrease in HF (n.u) by standing from the supine position were lower in diabetic patients compared with those in controls. CONCLUSION: The COMPASS score correlated with some component score of the HRV in diabetics. The HRV may be used as a tool to detect diabetic autonomic neuropathy by augmentation with position change.