Purusharth I. Rajyaguru

DDX3X is a highly conserved RNA helicase associated with RNA metabolism, translation initiation, and deciding cell fate choices. Spontaneous mutations in DDX3X cause a rare genetic human disorder called DDX3X syndrome, showing a spectrum of neurodevelopmental and intellectual abnormalities. How missense mutations in DDX3X lead to aberrant cellular functions and pathological consequences is unclear. Here, we demonstrate that specific DDX3X syndrome missense mutations induce the formation of persistent, solid-like DDX3X stress granules by structurally altering the conformation of the DDX3X-RNA complex. Structural interrogation of DDX3X syndrome missense mutations revealed critical mutations that perturb the DDX3X-RNA complex, exhibit high clinical pathogenicity scores, and are associated with cancers. N and C-terminal mutations adjacent to the helicase domain of the DDX3X (F182V, I190S, T198P, L556S, and L559H) showed augmented stress granule (SG) assembly, and formation of persistent DDX3X-SGs in neuronal and non-neuronal cells. Intriguingly, these mutations altered the liquid-like properties of DDX3X-SGs, forming solid-like SGs. Mechanistically, these mutations drive the persistent DDX3X-SGs by either locking DDX3X in an open, RNA-bound conformation or by increasing the rigidity of the DDX3X-RNA complex (I190S; T198P), thereby conferring a loss of liquid-like properties. The persistent solid-like DDX3X-SGs preferentially promoted neuronal lytic cell death and did not affect translation. Also, the C-terminal L556S and L559H mutations, which form persistent granules, promoted the DDX3X-driven β-amyloid aggregation. Our observations indicate that DDX3X syndrome mutations near the N- and C-termini promote the formation of solid-like DDX3X condensates, neuropathological aggregation, and neuronal cell death, which might underlie the disease pathogenesis in humans.

The fine balance between cellular homeostasis and stress response is crucial for cell survival under conditions of genotoxic stress. Here, we identify a regulatory role for the translation repressor Sbp1 in modulating autophagy during hydroxyurea (HU)-induced replication stress. We observe that Sbp1 localizes to reversible, mRNA-containing cytoplasmic granules specifically upon HU treatment in an RGG motif-dependent manner. Loss of Sbp1 leads to selective translational upregulation of key autophagy genes ATG1, ATG2, and ATG9. Consistent with these translational changes, sbp1∆ cells exhibit increased selective macroautophagy/autophagy and enhanced bulk autophagy, whereas Sbp1 overexpression suppresses both processes. Interestingly, overexpression of Sbp1 shifts DNA repair toward non-homologous end joining (NHEJ) repair, linking altered autophagy to genome maintenance. Together, these findings identify Sbp1 as a negative regulator of autophagy during replication stress and suggest a regulatory axis linking granule-mediated mRNA sequestration, translational control of autophagy factors, and the cellular response to genotoxic stress.Abbreviations: CHX: cycloheximide; CPT: camptothecin; DDR: DNA damage response; GTA: genotoxin-associated targeted autophagy; HR: homologous recombination; HU: hydroxyurea; MMS: methyl methanesulfonate; mRNPs: mRNA-protein complexes; NHEJ: non-homologous end joining; P-bodies: processing bodies; RBPs: RNA binding proteins.

Protein homeostasis, the balance between protein folding, function, and clearance, is essential for cellular health, and its disruption is a hallmark of many neurodegenerative diseases, including Parkinson’s disease. Arginine methylation, carried out by enzymes like Hmt1 in yeast and PRMT1 in humans, is best known for regulating transcription, translation, apoptosis and pre-mRNA splicing. In this study, we reveal a surprising, non-canonical role for these methyltransferases in controlling the aggregation and toxicity of α-synuclein, a protein lacking arginine and implicated in Parkinson’s disease pathology. We find that under oxidative stress, Hmt1 and PRMT1 relocate from the nucleus into the cytoplasm. Using genetic, biochemical and imaging assays in yeast and mammalian cells, we observe that Hmt1/PRMT1 dissolve aggregates, promotes their removal through the ubiquitin–proteasome system, and reduces α-synuclein toxicity. Importantly, this activity does not require the classical methyltransferase function. Catalytically inactive mutants and a specific PRMT1 inhibitor both enhance aggregate clearance and improve cell survival. Together, our findings uncover an unexpected function for Hmt1 and PRMT1 in maintaining protein quality under stress, expanding our understanding of how cells cope with toxic protein assemblies and pointing to new directions for therapeutic strategies in protein aggregation disorders.

In response to stress, cells undergo gene expression reprogramming to cope with external stimuli. Cells utilize a conserved stress response mechanism called global downregulation of translation, leading to the storage of translationally repressed mRNAs in RNA granules. During oxidative stress induced by H2O2, genes responsible for combating oxidative stress, such as catalases, are strongly induced. However, the post-transcriptional regulatory events affecting these genes during H2O2 stress are not well-explored. Scd6, an RGG-motif-containing protein in yeast, acts as a translational repressor through its interaction with eIF4G1. This study identifies the role of Scd6 in oxidative stress response by regulating cytoplasmic catalase T1 (CTT1). We observe that peroxide stress induces the assembly of Scd6 puncta, which do not colocalize with P-bodies or stress granules. Scd6 overexpression increased sensitivity, while deletion enhanced tolerance to H2O2 treatment. Increased ROS accumulation and decreased Ctt1 protein levels were observed upon Scd6 overexpression due to translation repression of CTT1 mRNA. CTT1 mRNA interacts with Scd6. smFISH analysis and RNA immunoprecipitation studies reveal that localization of Scd6 to puncta upon peroxide stress reduces its interaction with CTT1 mRNA, allowing derepression. The role of Scd6 in peroxide stress response is conserved since the human homolog LSm14A also localizes to puncta upon H2O2 stress, and its overexpression reduces survival in response to peroxide stress. Overall, this study identifies a unique example of translation regulation whereby stress-induced localization of the translation repressor protein to puncta leads to derepression of the target mRNA.

Scientific advances have emboldened human efforts toward exploring the potential of extinct, extant, or future life on Mars. An important aspect of this endeavor is understanding how an organism adapts to stress-inducing environmental conditions on Mars, such as radiation, shock waves, extreme temperatures, and chaotropic stress due to higher levels of perchlorates. A conserved approach used by organisms across evolutionary scales to adapt and overcome stress conditions is the assembly of ribonucleoprotein (RNP) condensates. In this study, we employ a multidisciplinary approach to understand yeast survivability and adaptation under Mars-like stress conditions, specifically shock waves and perchlorate, by focusing on RNP condensates. Our study reveals that yeast survives 5.6 M intensity shock waves. Exposure to either shock waves or sodium perchlorate induces the formation of P-bodies, a conserved stress-induced condensate. Yeast mutants defective in P-body assembly show defective growth in response to perchlorate stress. Transcriptome analysis, followed by validation, identified several relevant transcripts whose levels are perturbed in response to Mars-like conditions. Finally, identification of several transcripts whose abundance is altered in the P-body assembly mutant upon stress highlights a new connection between response to Martian stress conditions and RNP condensates. This study, a first of its kind, highlights the importance of RNP condensates in understanding the impact of Martian conditions on life in general. This study paves the way for using RNP condensates as a biomarker for assessing the health of life forms during space explorations.

Scientific advances have emboldened human efforts toward exploring the potential of extinct, extant, or future life on Mars. An important aspect of this endeavor is understanding how an organism adapts to stress-inducing environmental conditions on Mars, such as radiation, shock waves, extreme temperatures, and chaotropic stress due to higher levels of perchlorates. A conserved approach used by organisms across evolutionary scales to adapt and overcome stress conditions is the assembly of ribonucleoprotein (RNP) condensates. In this study, we employ a multidisciplinary approach to understand yeast survivability and adaptation under Mars-like stress conditions, specifically shock waves and perchlorate, by focusing on RNP condensates. Our study reveals that yeast survives 5.6 M intensity shock waves. Exposure to either shock waves or sodium perchlorate induces the formation of P-bodies, a conserved stress-induced condensate. Yeast mutants defective in P-body assembly show defective growth in response to perchlorate stress. Transcriptome analysis, followed by validation, identified several relevant transcripts whose levels are perturbed in response to Mars-like conditions. Finally, identification of several transcripts whose abundance is altered in the P-body assembly mutant upon stress highlights a new connection between response to Martian stress conditions and RNP condensates. This study, a first of its kind, highlights the importance of RNP condensates in understanding the impact of Martian conditions on life in general. This study paves the way for using RNP condensates as a biomarker for assessing the health of life forms during space explorations.