Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid ß-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid ß-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid ß-oxidation.

Interplay of RNA-binding proteins controls germ cell development in zebrafish.

The specification of germ cells in zebrafish mostly relies on an inherited mechanism by which localized maternal determinants, called germ plasm, confer germline fate in the early embryo. Extensive studies have partially allowed the identification of key regulators governing germ plasm formation and subsequent germ cell development. RNA-binding proteins, acting in concert with other germ plasm components, play essential roles in the organization of the germ plasm and the specification, migration, maintenance, and differentiation of primordial germ cells. The loss of their functions impairs germ cell formation and causes sterility or sexual conversion. Evidence is emerging that they instruct germline development through differential regulation of mRNA fates in somatic and germ cells. However, the challenge remains to decipher the complex interplay of maternal germ plasm components in germ plasm compartmentalization and germ cell specification. Since failure to control the developmental outcome of germ cells disrupts the formation of gametes, it is important to gain a complete picture of regulatory mechanisms operating in the germ cell lineage. This review sheds light on the contributions of RNA-binding proteins to germ cell development in zebrafish and highlights intriguing questions that remain open for future investigation.

The H1/H5 domain contributes to OsTRBF2 phase separation and gene repression during rice development.

Transcription factors (TFs) tightly control plant development by regulating gene expression. The phase separation of TFs plays a vital role in gene regulation. Many plant TFs have the potential to form phase-separated protein condensates; however, little is known about which TFs are regulated by phase separation and how it affects their roles in plant development. Here, we report that the rice (Oryza sativa) single Myb TF TELOMERE REPEAT-BINDING FACTOR 2 (TRBF2) is highly expressed in fast-growing tissues at the seedling stage. TRBF2 is a transcriptional repressor that binds to the transcriptional start site of thousands of genes. Mutation of TRBF2 leads to pleiotropic developmental defects and misexpression of many genes. TRBF2 displays characteristics consistent with phase separation in vivo and forms phase-separated condensates in vitro. The H1/H5 domain of TRBF2 plays a crucial role in phase separation, chromatin targeting and gene repression. Replacing the H1/H5 domain by a phase-separated intrinsically disordered region from Arabidopsis (Arabidopsis thaliana) AtSERRATE partially recovers the function of TRBF2 in gene repression in vitro and in transgenic plants. We also found that TRBF2 is required for trimethylation of histone H3 Lys27 (H3K27me3) deposition at specific genes and genome-wide. Our findings reveal that phase separation of TRBF2 facilitates gene repression in rice development.

The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process.

N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

CaMKIV-Mediated Phosphorylation Inactivates Freud-1/CC2D1A Repression for Calcium-Dependent 5-HT1A Receptor Gene Induction.

Calcium calmodulin-dependent protein kinase (CaMK) mediates calcium-induced neural gene activation. CaMK also inhibits the non-syndromic intellectual disability gene, Freud-1/CC2D1A, a transcriptional repressor of human serotonin-1A (5-HT1A) and dopamine-D2 receptor genes. The altered expression of these Freud-1-regulated genes is implicated in mental illnesses such as major depression and schizophrenia. We hypothesized that Freud-1 is blocked by CaMK-induced phosphorylation. The incubation of purified Freud-1 with either CaMKIIα or CaMKIV increased Freud-1 phosphorylation that was partly prevented in Freud-1-Ser644Ala and Freud-1-Thr780Ala CaMK site mutants. In human SK-N-SH neuroblastoma cells, active CaMKIV induced the serine and threonine phosphorylation of Freud-1, and specifically increased Freud-1-Thr780 phosphorylation in transfected HEK-293 cells. The activation of purified CaMKIIα or CaMKIV reduced Freud-1 binding to its DNA element on the 5-HT1A and dopamine-D2 receptor genes. In SK-N-SH cells, active CaMKIV but not CaMKIIα blocked the Freud-1 repressor activity, while Freud-1 Ser644Ala, Thr780Ala or dual mutants were resistant to inhibition by activated CaMKIV or calcium mobilization. These results indicate that the Freud-1 repressor activity is blocked by CaMKIV-induced phosphorylation at Thr780, resulting in the up-regulation of the target genes, such as the 5-HT1A receptor gene. The CaMKIV-mediated inhibition of Freud-1 provides a novel de-repression mechanism to induce 5-HT1A receptor expression for the regulation of cognitive development, behavior and antidepressant response.

Structure-activity relationship study of nitrogen signaling factors.

We have synthesized 10 analogs of oxylipins, which are nitrogen signaling factors (NSFs) that mediate cell-to-cell communication in the fission yeast Schizosaccharomyces pombe, and evaluated their structure-activity relationships with the aim of developing molecular probes for NSFs. We found that the OH or OAc group at C10 could be replaced with a compact amide (17) or carbamate (19). Introducing an alkyne as a detection tag at C10 led to decreased, though still sufficient, activity. Introducing an alkyne at the C18 position showed a similar trend, suggesting tolerance is relatively low even for compact functional groups such as alkynes. Although introduction of a diazirine moiety as a photoreactive group at the C5 position decreased the activity, we found that introducing diazirine at the C13 position was acceptable, and compound 38 exhibited potent NSF activity. These findings will be helpful in the development of molecular probes for NSFs.

Graded-CRISPRi, a Tool for Tuning the Strengths of CRISPRi-Mediated Knockdowns in Vibrio natriegens Using gRNA Libraries.

In recent years, the fast-growing bacterium Vibrio natriegens has gained increasing attention as it has the potential to become a next-generation chassis for synthetic biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression levels of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.

PPARα phosphorylation regulates colorectal tumor immune escape.

A high level of PD-L1 in cancer cells promotes tumor immune escape and inhibits tumor immunotherapy. Although PD-L1 gene expression is upregulated by multiple pathways, its gene transcriptional repression is still unclear. Here we found that loss of PPARα, one of the peroxisome-proliferator-activated receptors (PPARs) family members, promoted colorectal tumor immune escape. Mechanistically, PPARα directly bound to the PD-L1 promoter resulting in its gene transcriptional repression, which in turn increased T cell activity, and PPARα agonist enhanced this event. However, ERK induced PPARα-S12 phosphorylation leading to blockade of PPARα-mediated PD-L1 transcriptional repression, and the combination of ERK inhibitor with PPARα agonist significantly inhibited tumor immune escape. These findings suggest that the ERK-PPARα pathway inhibited PD-L1 gene transcriptional repression and promoted colorectal tumor immune escape.

Genome-wide analysis of TOPLESS/TOPLESS-RELATED co-repressors and functional characterization of BnaA9.TPL regulating the embryogenesis and leaf morphology in rapeseed.

TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins belong to the Groucho (Gro)/Tup1 family co-repressors and act as broad co-repressors that modulate multiple phytohormone signalling pathways and various developmental processes in plant. However, TPL/TPR co-repressors so far are poorly understood in the rapeseed, one of the world-wide important oilseed crops. In this study, we comprehensively characterized eighteen TPL/TPR genes into five groups in the rapeseed genome. Members of TPL/TPR1/TPR4 and TPR2/TPR3 had close evolutionary relationship, respectively. All TPL/TPRs had similar expression patterns and encode conserved protein domain. In addition, we demonstrated that BnaA9.TPL interacted with all known plant repression domain (RD) sequences, which were distributed in non-redundant 24,238 (22.6 %) genes and significantly enriched in transcription factors in the rapeseed genome. These transcription factors were largely co-expressed with the TPL/TPR genes and involved in diverse pathway, including phytohormone signal transduction, protein kinases and circadian rhythm. Furthermore, BnaA9.TPL was revealed to regulate apical embryonic fate by interaction with Bna.IAA12 and suppression of PLETHORA1/2. BnaA9.TPL was also identified to regulate leaf morphology by interaction with Bna.AS1 (Asymmetric leaves 1) and suppression of KNOTTED-like homeobox genes and YABBY5. These data not only suggest the rapeseed TPL/TPRs play broad roles in different processes, but also provide useful information to uncover more TPL/TPR-mediated control of plant development in rapeseed.

Identification success rates in the post-Spanish Civil War mass graves located in the cemetery of Paterna, Spain: Meta-research on 15 mass graves with 933 subjects.

Spain suffered a Civil War between 1936 and 1939 that ended with the victory of the National Forces led by General Franco. Once the Spanish Civil War ended, 2238 subjects were executed and buried in several mass graves in the Cemetery of Paterna, one of Spain's largest mass grave sites. Efforts to locate and identify all the victims of the mass graves of the Paterna cemetery are ongoing, but the actual data of the percentage of DNA identifications remains uncertain. Following this, we conducted a meta-research study including 15 mass graves and 933 subjects to determine the DNA identification rates in the mass graves of the Paterna cemetery. We found that the total proportion of identified subjects in the mass graves was 15.9 % (95 % CI: 10.0-22.9). Moreover, we found that the model between the identification success rate (ISR) and the number of relatives that donated DNA (NRTDD) in the mass graves of the cemetery of Paterna was ISR = NRTDD-0.424. Results obtained about the proportion of identified subjects and the model between the ISR and the NRTDD imply the need for a scientific reflection between all the research groups involved in the identification tasks to modify deficiencies and update identification protocols to obtain better future results.

Virtually identical does not mean exactly identical: Discrepancy in energy metabolism between glucose and fructose fermentation influences the reproductive potential of yeast cells.

The physiological efficiency of cells largely depends on the possibility of metabolic adaptations to changing conditions, especially on the availability of nutrients. Central carbon metabolism has an essential role in cellular function. In most cells is based on glucose, which is the primary energy source, provides the carbon skeleton for the biosynthesis of important cell macromolecules, and acts as a signaling molecule. The metabolic flux between pathways of carbon metabolism such as glycolysis, pentose phosphate pathway, and mitochondrial oxidative phosphorylation is dynamically adjusted by specific cellular economics responding to extracellular conditions and intracellular demands. Using Saccharomyces cerevisiae yeast cells and potentially similar fermentable carbon sources i.e. glucose and fructose we analyzed the parameters concerning the metabolic status of the cells and connected with them alteration in cell reproductive potential. Those parameters were related to the specific metabolic network: the hexose uptake - glycolysis and activity of the cAMP/PKA pathway - pentose phosphate pathway and biosynthetic capacities - the oxidative respiration and energy generation. The results showed that yeast cells growing in a fructose medium slightly increased metabolism redirection toward respiratory activity, which decreased pentose phosphate pathway activity and cellular biosynthetic capabilities. These differences between the fermentative metabolism of glucose and fructose, lead to long-term effects, manifested by changes in the maximum reproductive potential of cells.

Sevoflurane inhibits lung cancer development by promoting FUS1 transcription via downregulating IRF6.

Lung cancer is a major contributor to cancer deaths worldwide and is on the rise. Although surgical resection has been widely used as a standard of therapy for lung cancer patients, the relapse rate after surgery is high. It is still unclear whether there is a potential drug that can reduce the probability of post-surgical recurrence in lung cancer patients. We used five typical lung cancer cell lines as well as 41 lung cancer tissue samples and paracancer tissue samples to investigate the expression levels of IRF6 and FUS1. We also treated lung cancer cells (H322 and A549) with different concentrations of sevoflurane to study its influence on lung cancer cell tumorigenesis. Lentivirus-mediated gain-of-function studies of IRF6 and FUS1 were applied to validate the role of IRF6 and FUS1 in lung cancer. Next, we used short hairpin RNA-mediated loss of function of IRF6 and luciferase, ChIP assays to validate the regulatory role of IRF6 on FUS1. Our findings reported that IRF6 was up-regulated in lung cancer tissues, while FUS1 was down-regulated. Functional assays revealed that sevoflurane inhibits lung cancer development by downregulating IRF6 expression. Luciferase assay and ChIP-qPCR assay uncovered that IRF6 represses FUS1 transcriptional expression in lung cancer cells. We have shown that sevoflurane prevents lung cancer development by downregulating IRF6 to stimulate FUS1 transcription; indicating that sevoflurane can be used as the potential anesthetic drug in surgical resection to reduce post-operative tumor relapse in lung cancer patients.

Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes.

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity, and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we use multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc-repressed genes. Finally, non-Hox Pc targets are more proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not induce frequent clustering of multiple distant Pc genes.

Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals.

Carbon source is crucial for the cell growth and metabolism in microorganisms, and its utilization significantly affects the synthesis efficiency of target products in microbial cell factories. Compared with a single carbon source, co-utilizing carbon sources provide an alternative approach to optimize the utilization of different carbon sources for efficient biosynthesis of many chemicals with higher titer/yield/productivity. However, the efficiency of bioproduction is significantly limited by the sequential utilization of a preferred carbon source and secondary carbon sources, attributed to carbon catabolite repression (CCR). This review aimed to introduce the mechanisms of CCR and further focus on the summary of the strategies for co-utilization of carbon sources, including alleviation of CCR, engineering of the transport and metabolism of secondary carbon sources, compulsive co-utilization in single culture, co-utilization of carbon sources via co-culture, and evolutionary approaches. The findings of representative studies with a significant improvement in the bioproduction of chemicals via the co-utilization of carbon sources were discussed in this review. It suggested that by combining rational metabolic engineering and irrational evolutionary approaches, co-utilizing carbon sources can significantly contribute to the bioproduction of chemicals.

Repression of Acinetobacter baumannii DNA damage response requires DdrR-assisted binding of UmuDAb dimers to atypical SOS box.

The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

Non-transcriptional regulatory activity of SMAX1 and SMXL2 mediates karrikin-regulated seedling response to red light in Arabidopsis.

Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways. The transcriptional repressor proteins SUPPRESSOR OF MAX2 1 (SMAX1), SMAX1-like2 (SMXL2), and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or by inhibiting the activities of transcription factors. In this study, we characterized the non-transcriptional regulatory activities of SMXL proteins in Arabidopsis. We discovered that SMAX1 and SMXL2 with mutations in their ethylene-response factor-associated amphiphilic repression (EAR) motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 mutant. SMAX1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR 4 (PIF4) and PIF5 to enhance their protein stability by interacting with phytochrome B (phyB) and suppressing the association of phyB with PIF4 and PIF5. The karrikin-responsive genes were then identified by treatment with GR24ent-5DS, a GR24 analog showing karrikin activity. Interestingly, INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) expression was repressed by GR24ent-5DS treatment in a PIF4- and PIF5-dependent and EAR-independent manner, whereas KARRIKIN UPREGULATED F-BOX 1 (KUF1) expression was induced in a PIF4- and PIF5-independent and EAR-dependent manner. Furthermore, the non-transcriptional regulatory activity of SMAX1, which is independent of the EAR motif, had a global effect on gene expression. Taken together, these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.

Utilization of carbon catabolite repression for efficiently biotransformation of anthraquinone O-glucuronides by Streptomyces coeruleorubidus DM.

Carbon catabolite repression (CCR) is a highly conserved mechanism that regulates carbon source utilization in Streptomyces. CCR has a negative impact on secondary metabolite fermentation, both in industrial and research settings. In this study, CCR was observed in the daunorubicin (DNR)-producing strain Streptomyces coeruleorubidus DM, which was cultivated in high concentration of carbohydrates. Unexpectedly, DM exhibited a high ability for anthraquinone glucuronidation biotransformation under CCR conditions with a maximum bioconversion rate of 95% achieved at pH 6, 30°C for 24 h. The co-utilization of glucose and sucrose resulted in the highest biotransformation rate compared to other carbon source combinations. Three novel anthraquinone glucuronides were obtained, with purpurin-O-glucuronide showing significantly improved water solubility, antioxidant activity, and antibacterial bioactivity. Comparative transcript analysis revealed that glucose and sucrose utilization were significantly upregulated as DM cultivated under CCR condition, which strongly enhance the biosynthetic pathway of the precursors Uridine diphosphate glucuronic acid (UDPGA). Meanwhile, the carbon metabolic flux has significantly enhanced the fatty acid biosynthesis, the exhaust of acetyl coenzyme A may lead to the complete repression of the biosynthesis of DNR, Additionally, the efflux transporter genes were simultaneously downregulated, which may contribute to the anthraquinones intracellular glucuronidation. Overall, our findings demonstrate that utilizing CCR can be a valuable strategy for enhancing the biotransformation efficiency of anthraquinone O-glucuronides by DM. This approach has the potential to improve the bioavailability and therapeutic potential of these compounds, opening up new possibilities for their pharmaceutical applications.

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development.

Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAA and HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.

Mining novel gene targets for improving tolerance to furfural and acetic acid in Yarrowia lipolytica using whole-genome CRISPRi library.

Abundant renewable resource lignocellulosic biomass possesses tremendous potential for green biomanufacturing, while its efficient utilization by Yarrowia lipolytica, an attractive biochemical production host, is restricted since the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate. Given deficient understanding of inherent interactions between inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using clustered regularly interspaced short palindromic repeats interference library in Y. lipolytica, achieving tolerance to 0.35 % (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15 % (v/v) acetic acid. The tolerance mechanism might involve improvement of cell division and decrease of reactive oxygen species level. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for improving microbial tolerance to lignocellulose-derived inhibitors and revealing underlying mechanism.

PolyQ-expanded ataxin-2 aggregation impairs cellular processing-body homeostasis via sequestering the RNA helicase DDX6.

Ataxin-2 (Atx2) is a polyglutamine (polyQ) tract-containing RNA-binding protein, while its polyQ expansion may cause protein aggregation that is implicated in the pathogenesis of neurodegenerative diseases such as spinocerebellar ataxia type 2 (SCA2). However, the molecular mechanism underlying how Atx2 aggregation contributes to the proteinopathies remains elusive. Here, we investigated the influence of Atx2 aggregation on the assembly and functionality of cellular processing bodies (P-bodies) by using biochemical and fluorescence imaging approaches. We have revealed that polyQ-expanded (PQE) Atx2 sequesters the DEAD-box RNA helicase (DDX6), an essential component of P-bodies, into aggregates or puncta via some RNA sequences. The N-terminal like-Sm (LSm) domain of Atx2 (residues 82-184) and the C-terminal helicase domain of DDX6 are responsible for the interaction and specific sequestration. Moreover, sequestration of DDX6 may aggravate pre-mRNA mis-splicing, and interfere with the assembly of cellular P-bodies, releasing the endoribonuclease MARF1 that promotes mRNA decay and translational repression. Rescuing the DDX6 protein level can recover the assembly and functionality of P-bodies, preventing targeted mRNA from degradation. This study provides a line of evidence for sequestration of the P-body components and impairment of the P-body homeostasis in dysregulating RNA metabolism, which is implicated in the disease pathologies and a potential therapeutic target.