A single-copy knockin translating ribosome immunoprecipitation toolkit for tissue-specific profiling of actively translated mRNAs in . .

Here, we introduce a single-copy knockin translating ribosome immunoprecipitation (SKI TRIP) toolkit, a collection of strains engineered by CRISPR in which tissue-specific expression of FLAG-tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. Through in-depth characterization of the effects of the FLAG tag in animals in which endogenous RPL-22 has been tagged, we show that it incorporates into actively translating ribosomes and efficiently and cleanly pulls down cell-type-specific transcripts. Importantly, the presence of the tag does not impact overall mRNA translation, create bias in transcript use, or cause changes to fitness of the animal.

Dual inhibition of HSF1 and DYRK2 impedes cancer progression.

Preserving proteostasis is a major survival mechanism for cancer. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a key oncogenic kinase that directly activates the transcription factor heat-shock factor 1 (HSF1) and the 26S proteasome. Targeting DYRK2 has proven to be a tractable strategy to target cancers sensitive to proteotoxic stress; however, the development of HSF1 inhibitors remains in its infancy.

Hexosamine pathway activation improves memory but does not extend lifespan in mice.

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals.

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway.

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure.

Perspective: Modulating the integrated stress response to slow aging and ameliorate age-related pathology.

Healthy aging requires the coordination of numerous stress signaling pathways that converge on the protein homeostasis network. The Integrated Stress Response (ISR) is activated by diverse stimuli, leading to phosphorylation of the eukaryotic translation initiation factor elF2 in its α-subunit. Under replete conditions, elF2 orchestrates 5' cap-dependent mRNA translation and is thus responsible for general protein synthesis.

NHR-8 and P-glycoproteins uncouple xenobiotic resistance from longevity in chemosensory mutants.

Longevity is often associated with stress resistance, but whether they are causally linked is incompletely understood. Here we investigate chemosensory-defective mutants that are long-lived and stress resistant. We find that mutants in the intraflagellar transport protein gene were significantly protected from tunicamycin-induced ER stress.

The RATIOnal Role of Polyamines in Epidermal Differentiation.

Polyamines have been implicated in skin tumorigenesis; however, their role in epidermal homeostasis remains obscure. In a new article in the Journal of Investigative Dermatology, Rahim et al. (2021) report that keratinocyte differentiation requires a shift in polyamine ratios that is mediated by AMD1.

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging.

Muscle function relies on the precise architecture of dynamic contractile elements, which must be fine-tuned to maintain motility throughout life. Muscle is also plastic, and remodeled in response to stress, growth, neural and metabolic inputs. The conserved muscle-enriched microRNA, miR-1, regulates distinct aspects of muscle development, but whether it plays a role during aging is unknown.

N1-acetylspermidine is a determinant of hair follicle stem cell fate.

Stem cell differentiation is accompanied by increased mRNA translation. The rate of protein biosynthesis is influenced by the polyamines putrescine, spermidine and spermine, which are essential for cell growth and stem cell maintenance. However, the role of polyamines as endogenous effectors of stem cell fate and whether they act through translational control remains obscure.

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme.

Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation.

Protein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B.

Small-molecule inhibitors of human mitochondrial DNA transcription.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines.

Glutamine Metabolism Controls Stem Cell Fate Reversibility and Long-Term Maintenance in the Hair Follicle.

Stem cells reside in specialized niches that are critical for their function. Upon activation, hair follicle stem cells (HFSCs) exit their niche to generate the outer root sheath (ORS), but a subset of ORS progeny returns to the niche to resume an SC state. Mechanisms of this fate reversibility are unclear.

Hexosamine Pathway Activation Improves Protein Homeostasis through the Integrated Stress Response.

Activation of the hexosamine pathway (HP) through gain-of-function mutations in its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT-1) ameliorates proteotoxicity and increases lifespan in Caenorhabditis elegans. Here, we investigate the role of the HP in mammalian protein quality control. In mouse neuronal cells, elevation of HP activity led to phosphorylation of both PERK and eIF2α as well as downstream ATF4 activation, identifying the HP as a modulator of the integrated stress response (ISR).

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis.

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear.

The E3 ubiquitin ligase UBR5 interacts with the H/ACA ribonucleoprotein complex and regulates ribosomal RNA biogenesis in embryonic stem cells.

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity.

Unbiased Forward Genetic Screening with Chemical Mutagenesis to Uncover Drug-Target Interactions.

The steadily increasing throughput in next-generation sequencing technologies is revolutionizing a number of fields in biology. One application requiring massive parallel sequencing is forward genetic screening based on chemical mutagenesis. Such screens interrogate the entire genome in an entirely unbiased fashion and can be applied to a number of research questions.

Chaperoning junior faculty: Institutional support and guidance can relieve challenges for early-career group leaders and improve academic performance.

Young, independent group leaders in academia face a range of new tasks and challenges. We suggest various measures for host institutes to help them to resolve these challenges and to flourish. [Image: see text]

Emerging topics in C. elegans aging research: Transcriptional regulation, stress response and epigenetics.

Key discoveries in aging research have been made possible with the use of model organisms. Caenorhabditis elegans is a short-lived nematode that has become a well-established system to study aging. The practicality and powerful genetic manipulations associated with this metazoan have revolutionized our ability to understand how organisms age.

Improved protein-crystal identification by using 2,2,2-trichloroethanol as a fluorescence enhancer.

The identification of initial lead conditions for successful protein crystallization is crucial for structural studies using X-ray crystallography. In order to reduce the number of false-negative conditions, an emerging number of fluorescence-based methods have been developed which allow more efficient identification of protein crystals and help to distinguish them from salt crystals. Detection of the native tryptophan fluorescence of protein crystals is one of the most widely used methods.

Unbiased compound-protein interface mapping and prediction of chemoresistance loci through forward genetics in haploid stem cells.

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes.

Oral Supplementation of Glucosamine Fails to Alleviate Acute Kidney Injury in Renal Ischemia-Reperfusion Damage.

Acute kidney injury is a leading contributor to morbidity and mortality in the ageing population. Proteotoxic stress response pathways have been suggested to contribute to the development of acute renal injury. Recent evidence suggests that increased synthesis of N-glycan precursors in the hexosamine pathway as well as feeding of animals with aminosugars produced in the hexosamine pathway may increase stress resistance through reducing proteotoxic stress and alleviate pathology in model organisms.