Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

GLTSCR2/PICT1 links mitochondrial stress and Myc signaling.

Mitochondrial defects underlie a multitude of human diseases. Genetic manipulation of mitochondrial regulatory pathways represents a potential therapeutic approach. We have carried out a high-throughput overexpression screen for genes that affect mitochondrial abundance or activity using flow-cytometry-based enrichment of a cell population expressing a high-complexity, concentration-normalized pool of human ORFs. The screen identified 94 candidate mitochondrial regulators including the nuclear protein GLTSCR2, also known as PICT1. GLTSCR2 enhances mitochondrial function and is required for the maintenance of oxygen consumption, consistent with a pivotal role in the control of cellular respiration. RNAi inactivation of the Caenorhabditis elegans ortholog of GLTSCR2 reduces respiration in worms, indicating functional conservation across species. GLTSCR2 controls cellular proliferation and metabolism via the transcription factor Myc, and is induced by mitochondrial stress, suggesting it may constitute a significant component of the mitochondrial signaling pathway.

CALITAS: A CRISPR-Cas-aware ALigner for In silico off-TArget Search.

We describe CALITAS, a CRISPR-Cas-aware aligner and integrated off-target search algorithm. CALITAS uses a modified and CRISPR-tuned version of the Needleman-Wunsch algorithm. It supports an unlimited number of mismatches and gaps and allows protospacer adjacent motif (PAM) mismatches or PAMless searches. CALITAS also includes an exhaustive search routine to scan genomes and genome variants provided with a standard Variant Call Format file. By default, CALITAS returns a single best alignment for a given off-target site, which is a significant improvement compared to other off-target algorithms, and it enables off-targets to be referenced directly using alignment coordinates. We validate and compare CALITAS using a selected set of target sites, as well as experimentally derived specificity data sets. In summary, CALITAS is a new tool for precise and relevant alignments and identification of candidate off-target sites across a genome. We believe it is the state of the art for CRISPR-Cas specificity assessments.

Defective brown adipose tissue thermogenesis and impaired glucose metabolism in mice lacking Letmd1.

Manipulation of energy-dissipating adipocytes has the potential to produce metabolic benefits. To this end, it is valuable to understand the mechanisms controlling the generation and function of thermogenic fat. Here, we identify Letm1 domain containing 1 (Letmd1) as a regulator of brown fat formation and function. The expression of Letmd1 is induced in brown fat by cold exposure and by ß-adrenergic activation. Letmd1-deficient mice exhibit severe cold intolerance concomitant with abnormal brown fat morphology, reduced thermogenic gene expression, and low mitochondrial content. The null mice exhibit impaired ß3-adrenoreceptor-dependent thermogenesis and are prone to diet-induced obesity and defective glucose disposal. Letmd1 was previously described as a mitochondrial protein, and we find that it also localizes to the nucleus and interacts with the transcriptional coregulator and chromatin remodeler Brg1/Smarca4, thus providing a way to impact thermogenic gene expression. Our study uncovers a role for Letmd1 as a key regulatory component of adaptive thermogenesis.

Dietary Restriction Extends Lifespan through Metabolic Regulation of Innate Immunity.

Chronic inflammation predisposes to aging-associated disease, but it is unknown whether immunity regulation might be important for extending healthy lifespan. Here we show that in C. elegans, dietary restriction (DR) extends lifespan by modulating a conserved innate immunity pathway that is regulated by p38 signaling and the transcription factor ATF-7. Longevity from DR depends upon p38-ATF-7 immunity being intact but downregulated to a basal level. p38-ATF-7 immunity accelerates aging when hyperactive, influences lifespan independently of pathogen exposure, and is activated by nutrients independently of mTORC1, a major DR mediator. Longevity from reduced insulin/IGF-1 signaling (rIIS) also involves p38-ATF-7 downregulation, with signals from DAF-16/FOXO reducing food intake. We conclude that p38-ATF-7 is an immunometabolic pathway that senses bacterial and nutrient signals, that immunity modulation is critical for DR, and that DAF-16/FOXO couples appetite to growth regulation. These conserved mechanisms may influence aging in more complex organisms.

The effects of hemsball on the motor proficiency of students with intellectual disabilities.

Objective: The effects of hemsball game skills development program on the motor proficiency level of children in the 12-16 age group with mild and moderate intellectual disabilities (ID) have been examined in this study. Method: A total of 50 (25 experimental group +25 control group) students, 23 of which had mild intellectual disabilities (ID) and 27 with moderate ID studying in special education classes in the schools in Afyonkarahisar province in Turkey participated in the study. While no training was given to the control group, the students in the experimental group were subjected to 60 min of applications involving a basic hemsball training program per day for 3 days per week throughout 12 weeks. Bruininks-Oseretsky Test of Motor Proficiency (BOT-2) sub-tests (balance, bilateral coordination, upper-limb coordination) were applied twice, once before starting the program (pre-test) and once after the program was completed (post-test). The obtained data were tested with paired-samples t-test and independent-samples t-test. Result and Conclusion: As a result of the study, it was noted that the application of the hemsball game skill development program had incurred significant differences between the experimental and control group according to the post-test as well as the pre-test and post-test for the experimental group which were positive. However, it was determined that the application was more effective in students with moderate level ID than students with mild ID.

MicroRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans.

Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.

SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans.

The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.

Expression pattern analysis of microRNAs in Caenorhabditis elegans.

MicroRNAs (miRNAs) are ∼22 nucleotide single-stranded RNA molecules that originate from hairpin precursors and regulate gene expression at the posttranscriptional level by basepairing with target messenger RNA and blocking its translation or inducing its degradation. miRNAs play important roles in a variety of biological processes, including development, proliferation, differentiation, cell fate determination, apoptosis, signal transduction, host-viral interactions, and tumorigenesis. Methodological advances in miRNA studies allowed identification of biological roles for many miRNAs, and establishing the spatiotemporal expression patterns of miRNAs is one of the approaches to elucidate their biological functions. Expression pattern analysis of miRNAs helps to identify potential genetic interactors that exhibit similar expression patterns and this, combined with further supporting experiments, helps to identify the genetic pathways in which the specific miRNAs are involved. In this chapter, we describe a detailed protocol for the analysis of miRNA expression patterns in Caenorhabditis elegans.

Biolistic transformation of Caenorhabditis elegans.

The ability to generate transgenic animals to study gene expression and function is a powerful and important part of the Caenorhabditis elegans genetic toolbox. Transgenic animals can be created by introducing exogenous DNA into the worm germline either by microinjection or by microparticle bombardment (biolistic transformation). In this chapter we describe a simple and robust protocol to generate transgenic C. elegans animals by biolistic transformation with gold particles using the Bio-Rad PDS-1000/He system with Hepta adapter and unc-119 selection marker. We also point out the steps that need special attention to achieve successful transformations.

Expression patterns of intronic microRNAs in Caenorhabditis elegans.

BACKGROUND: MicroRNAs (miRNA) are an abundant and ubiquitous class of small RNAs that play prominent roles in gene regulation. A significant fraction of miRNA genes reside in the introns of the host genes in the same orientation and are thought to be co-processed from the host gene mRNAs and thus depend on the host gene promoter for their expression. However, several lines of evidence for independent expression of intronic miRNAs exist in the literature but the extent of this independence remains unclear. RESULTS: We performed a systematic analysis of genomic regions surrounding intronic miRNAs in the nematode Caenorhabditis elegans and found that, in many cases, there are extended intronic sequences immediately upstream of the miRNAs that are well-conserved between the nematodes. We have generated transcriptional green fluorescent protein reporter fusions in transgenic C. elegans lines and demonstrated that, in all seven investigated cases, the conserved sequences show promoter properties and produce specific expression patterns that are different from the host gene expression patterns. The observed expression patterns are corroborated by the published small RNA sequencing data. CONCLUSIONS: Our analysis reveals that the number of intronic miRNAs that do not rely on their host genes for expression is substantially higher than previously appreciated. At least one-third of the same-strand intronic miRNAs in C. elegans posses their own promoters and, thus, could be transcribed independently from their host genes. These findings provide a new insight into the regulation of miRNA genes and will be useful for the analysis of interactions between miRNAs and their host genes.