bims-ribost 2025-12-28 papers

bims-ribost

Biomed News

on Ribostasis and translation stress

Issue of 2025–12–28
29 papers selected by
Cédric Chaveroux, CNRS

eIF3: a critical player in mRNA recruitment to the ribosome with emerging roles across translation.
Current Knowledge of the Integrated Stress Response in the Development and Management of Acute Myeloid Leukemia: A Novel Target with Encouraging Progress.
Integrated stress response inhibition prolongs the lifespan of a Pelizaeus-Merzbacher disease mouse model by increasing oligodendrocyte survival.
Lipid Transfer Proteins and PI4KIIα Generate a Phosphoinositide-Linked Proteome.
Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation.
Conserved RNA helicase Vasa regulates ribonucleoprotein condensates through protein interaction and mRNA recruitment.
snoCLASH Reveals Extensive snoRNA-mRNA Interaction Networks.
Developing Topics.
Insights into PINK1/Parkin function and dysfunction from Drosophila models.
Trifloxystrobin induces oxidative stress-dependent activation of the OMA1-DELE1-HRI integrated stress response leading to apoptosis in human neuroblastoma cells.
Basic Science and Pathogenesis.
Basic Science and Pathogenesis.
Intracellular aggregation of the transthyretin protein is limited by Hsp70 chaperones.
Suboptimal codon pairs trigger ribosome collisions and cellular quality control responses in tRNA modification mutants.
Defining substrate specificities of human RNA capping methyltransferases through quantitative assessment of independent yet cooperative activities.
Uncovering the regulatory network of the small RNA SuhB and its contribution to stress resistance in Sphingopyxis granuli TFA.
Identification of myokines associated with the pathological stress response in the mdx mouse model of Duchenne muscular dystrophy.
Estimation of Telomeric Repeat-containing RNA from DNA/RNA Hybrid Complexes.
Mechanistic Investigation of Adociaquinone and Xestoquinone Derivatives in Breast Cancer Cells.
An RBPMS-driven splicing regulatory axis including MBNL1, RBFOX2, and QK promotes smooth muscle cell contractile identity.
Vaccinia virus mRNAs containing long 5'-poly(A)-leaders lack a canonical 5'-methylguanosine cap.
Multiple checkpoints ensure ribosomes have the correct end.
Azidohomoalanine (AHA) Metabolic Labeling Reveals Unique Proteomic Insights into Protein Synthesis and Degradation in Response to Bortezomib Treatment.
Emerging roles of stress granules in liver diseases: a comprehensive review.
Five hundred million years of hunger: how animals evolved to survive essential amino acid scarcity.
Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.
Molecular profiling of glioblastoma-derived extracellular vesicles identifies small nucleolar RNAs as candidate liquid biomarkers for radiation-induced senescence.
Purification and characterization of recombinant human translation initiation factor eIF3.
Pseudouridine increases ribosome stability in a thermophilic eukaryote.

Biochem Soc Trans. 2025 Dec 24. 53(6): 1527-1541

eIF3: a critical player in mRNA recruitment to the ribosome with emerging roles across translation.

Nicholas A Ide, Sufana Noorwez, Christine Xu, Colin Echeverría Aitken.

  Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that plays critical roles throughout translation initiation and has been implicated in a variety of human diseases. More recently, eIF3 has been tied to translation elongation and termination, as well as translational regulation. And yet, a mechanistic understanding of how eIF3 and its constituent subunits perform their canonical roles during initiation continues to elude us. Work across the last two decades has delineated broad mechanistic roles for some of these subunits and identified distinct modules of the complex that contribute differentially to the recruitment of messenger RNA (mRNA) to the ribosome during initiation. Structural approaches have further illuminated these putative roles. And yet, key mechanistic questions tied to fundamental technical challenges remain. Even so, new developments are poised to address these challenges and push our understanding of eIF3 function forward in the coming years.

Keywords:  MRNA; biochemistry; eukaryotic gene expression; molecular mechanisms; ribosomes; translation

DOI:  https://doi.org/10.1042/BST20253069
Drug Des Devel Ther. 2025 ;19 11269-11288

Current Knowledge of the Integrated Stress Response in the Development and Management of Acute Myeloid Leukemia: A Novel Target with Encouraging Progress.

Wanzhi Jiang, Yaonan Hong, Peicheng Wang, Xiawan Yang, Keding Shao, Man Li, Dijiong Wu.

  Primary or acquired resistance to standard chemotherapy and novel targeted therapies remains a common cause of relapsed/refractory acute myeloid leukemia (AML). The five-year overall survival rate for AML patients remains poor. Exploring novel therapeutic pathways may offer effective strategies to address this challenge. The Integrated Stress Response (ISR) is a signaling pathway that maintains cellular homeostasis by reducing global protein synthesis in response to external and internal stressors. Recent studies have demonstrated that ISR exerts a dual role in AML. Moderate activation of ISR supports hematopoietic and leukemia stem cell maintenance and promotes AML progression, whereas hyperactivation of ISR induces apoptosis and reduces myeloid cell leukemia-1 (MCL-1) expression. MCL-1 overexpression contributes to venetoclax resistance. However, MCL-1 inhibitors have shown disappointing cardiac toxicity in clinical studies. Hyperactivation of the ISR can indirectly suppress MCL-1 and help reverse venetoclax (ABT-199) resistance, as reported in previous studies. Our previous study also indicates that ISR activation can reverse venetoclax resistance in AML cells. These findings support the ISR as a novel therapeutic target in AML. However, the mechanisms by which ISR influences stemness and resistance are not yet fully understood. This review integrates current mechanistic insights and preclinical evidence to highlight the ISR as both a key driver of leukemogenesis and a promising target for overcoming drug resistance in AML. We searched the literature up to October 2025 in PubMed, Google Scholar, and ClinicalTrials.gov using terms related to AML, ISR signaling, venetoclax, and ISR kinases.

Keywords:  ATF4; acute myeloid leukemia; eIF2α; integrated stress response; resistance

DOI:  https://doi.org/10.2147/DDDT.S573043
Nat Commun. 2025 Dec 26.

Integrated stress response inhibition prolongs the lifespan of a Pelizaeus-Merzbacher disease mouse model by increasing oligodendrocyte survival.

Yanan Chen, Rejani B Kunjamma, Karin Lin, Li Kai, Maria Dima, Kody Bruce, Ian Steckler, Young Hyun Che, Jason Chan-Zervas, Jaime Eugenin von Bernhardi, Caitlin Connelly, Sude Ece, Grace Newell, Dwight E Bergles, Carmela Sidrauski, Brian Popko.

The leukodystrophy Pelizaeus-Merzbacher disease (PMD) is caused by myelin protein proteolipid protein gene (PLP1) mutations. PMD is characterized by oligodendrocyte death and CNS hypomyelination; thus, increasing oligodendrocyte survival and enhancing myelination could provide therapeutic benefit. Here, we use the PMD mouse model Jimpy to determine the impact of the integrated stress response (ISR) on the oligodendrocyte response to mutant PLP expression. Male Jimpy animals in which the ISR-triggering eukaryotic initiation factor (eIF) 2α kinase, protein kinase-like endoplasmic reticulum kinase (PERK), is inactivated have an extended lifespan that correlates with increased oligodendrocyte survival and enhanced CNS myelination. Inactivation of downstream components of the ISR pathway, in contrast, does not rescue oligodendrocytes or myelin. Phosphorylated eIF2α inhibits the exchange factor eIF2B, resulting in diminished protein synthesis. Treatment with small molecule eIF2B activators 2BAct and ISRIB increases oligodendrocyte survival, CNS myelination, and doubled the Jimpy lifespan. These results suggest that ISR modulation could provide therapeutic benefit to PMD patients.

DOI: https://doi.org/10.1038/s41467-025-68045-0
bioRxiv. 2025 Dec 18. pii: 2025.12.17.694959. [Epub ahead of print]

Lipid Transfer Proteins and PI4KIIα Generate a Phosphoinositide-Linked Proteome.

Noah D Carrillo, Mo Chen, Poorwa Awasthi, Tianmu Wen, Xiangqin Chen, Giang Vu, Dhruv Brahmbhatt, Michael Herlihy, Benjamin B Minkoff, Michael Sussman, Vincent L Cryns, Richard A Anderson.

Phosphoinositide (PIP n ) lipid second messengers in membranes regulate a myriad of cellular processes. In the cytosol, the phosphatidylinositol (PI) 3-kinase (PI3K)/Akt pathway is scaffolded on IQGAP1 to facilitate the activation of Akt by the synthesis of PI3,4,5P 3 . In the nucleus, PIP n signaling occurs in regions devoid of membranes via their stable association with proteins. While several of these proteins have been identified, understanding the extent and impact of protein-linked PIP n signaling warrants further investigation. The tumor suppressor p53, was shown in the companion paper to be regulated by PI transfer proteins (PITPs) and a PI 4-kinase (PI4KIIα), which are required to form p53-PIP n complexes that assemble a nuclear PI3K/Akt pathway. Here we report that class I PITPs (PITPα/β) and PI 4-kinase initiate PIP n linkages to many different proteins. PITPα/β and PI4KIIα accumulate in the nucleoplasm in response to stress and are necessary to synthesize nuclear PIP n s linked to proteins. These PITPα/β-dependent protein-PIP n complexes are detected by metabolically labeling cells with the PIP n precursor [H 3 ]- myo -inositol and resist denaturation and SDS-PAGE, indicating that these protein-PIP n complexes represent a putative posttranslational modification. Proteomic analyses of proteins that are regulated by PITPα/β and/or are linked to PI4,5P 2 have identified an emerging PIPylome that is enriched in metabolic, signaling, cytoskeletal and DNA repair pathway components. Taken together, these data provide evidence for an emerging proteome with linked PIP n s that represent a PIP n signaling paradigm that is distinct from the membrane-localized pathway but utilizes many of the same PIP kinases and phosphatases.
In brief: Phosphatidylinositol transfer proteins and PI 4-kinase initiate a PIP n -linked protein network in membrane-free regions.

DOI: https://doi.org/10.64898/2025.12.17.694959
bioRxiv. 2025 Dec 08. pii: 2025.12.08.692954. [Epub ahead of print]

Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation.

Dorthy Fang, John M Babich, Ryan Stanton, Isaac W Vock, Kyrillos Abdallah, Mingyi Zhu, Richard Li, Matthew D Simon, Wendy V Gilbert, Sigrid Nachtergaele.

Despite recent advances in technology to map RNA chemical modifications transcriptome-wide, the distribution of N¹-methyladenosine (m¹A) in mRNA remains contested, hindering a clear understanding of its function. Additionally, the enzyme(s) that installs the majority of reported mRNA m 1 A sites has yet to be identified. In this study, we characterized TRMT61B, an m 1 A methyltransferase known to methylate mitochondrial RNAs, but whose sequence preferences have been underexplored. By integrating cellular overexpression of TRMT61B and in vitro methylation of a synthetic pool of diverse human RNA sequences, we identified a preference for a YMR A consensus motif in single stranded RNA regions. In these experiments, TRMT61B methylated thousands of novel human mRNA sites, revealing activity on cytosolic mRNAs. We used these novel m 1 A-modifiable sequences to test the effects of m 1 A on translation of luciferase reporters and on ribosome recruitment to modified transcripts in the pool. We found that m 1 A addition can significantly affect translation and ribosome recruitment, but that these effects are vary by transcript. Taken together, our results can inform future studies of TRMT61B and mRNA, and emphasize that studies of m 1 A regulation of mRNA must be carried out and interpreted in a highly context-aware manner.

DOI: https://doi.org/10.64898/2025.12.08.692954
bioRxiv. 2025 Dec 20. pii: 2025.12.19.695503. [Epub ahead of print]

Conserved RNA helicase Vasa regulates ribonucleoprotein condensates through protein interaction and mRNA recruitment.

Siran Tian, Hyosik Kim, Harrison A Curnutte, Tatjana Trcek.

Germlines contain ribonucleoprotein condensates known as germ granules, which concentrate proteins and mRNAs essential for animal development. Vasa, a conserved DEAD-box RNA helicase, is a core and highly concentrated constituent of germ granules. However, its roles within these structures remain poorly understood. Here, we use Drosophila germ granules as a model system to address this question. Applying in vivo and human cell systems, we found that condensation of Oskar (Osk) protein, which nucleates germ granules, occurred independently of Vasa. However, in oocytes lacking detectable Vasa protein, Osk formed aggregated condensates regardless of the eGFP tag. Furthermore, Osk-eGFP showed minimal recovery as measured by fluorescence recovery after photobleaching (FRAP) indicating that its exchange between condensates and their surroundings was greatly reduced in the absence of Vasa. Supporting this result, co-expression of Vasa increased the FRAP recovery of Osk-eGFP condensates and reduced Osk-eGFP partitioning into them in cells. This effect required the interaction between Vasa and Osk, suggesting that by binding Osk, Vasa modulates Osk phase behavior and its condensate material properties. Super-resolution microscopy further revealed that Vasa is required for germ granule mRNA localization to Osk condensates in vivo. Co-expression of Vasa with Osk-eGFP is necessary and sufficient to recruit germ granule mRNAs to condensates in cells. Although this activity depends on Vasa-Osk interaction, the interaction itself is not sufficient. Notably, localization of a conserved germ granule mRNA nanos reduced the FRAP recovery of Osk-eGFP condensates in cells, partially counteracting Vasa effect. Collectively, our findings uncovered a novel function of the DEAD-box RNA helicase Vasa in regulating the material properties of Osk condensates through coordinated protein-protein and protein-mRNA interactions.

DOI: https://doi.org/10.64898/2025.12.19.695503
bioRxiv. 2025 Dec 11. pii: 2025.12.10.693487. [Epub ahead of print]

snoCLASH Reveals Extensive snoRNA-mRNA Interaction Networks.

Brittany A Elliott, Gene Yang, Alex K Choi, Yinzhou Zhu, Wesley R Freeman, Christopher L Holley.

Small nucleolar RNAs (snoRNAs) are classically defined as guides for ribosomal RNA (rRNA) modification, yet increasing evidence suggests that box C/D snoRNAs also interact with non-rRNA transcripts. Systematic discovery of such interactions has been hindered by overwhelming rRNA abundance and technical limitations in RNA-RNA capture. Here, we present snoCLASH, an optimized snoRNA RNA binding protein (RBP)-based crosslinking, ligation, and sequencing framework that integrates phenol-toluol extraction, polyA enrichment, nuclear fractionation, rRNA depletion, and dual-reference chimeric read analysis to enable transcriptome-scale identification of snoRNA-non-rRNA interactions. Applying this approach reveals thousands of snoRNA-associated mRNA regions spanning coding and regulatory elements and enriched for RBPs linked to epitranscriptomic regulation. Using this framework, we identify high-confidence snoRNA-mRNA interactions and functionally validate one candidate, demonstrating that a snoCLASH-discovered target undergoes snoRNA-dependent 2'-O-methylation with downstream effects on protein expression. Together, this work establishes snoCLASH as a scalable platform for discovering and validating non-canonical snoRNA targets beyond the ribosome.

DOI: https://doi.org/10.64898/2025.12.10.693487
Alzheimers Dement. 2025 Dec;21 Suppl 7 e108383

Developing Topics.

Hui Zheng.

BACKGROUND: Lysosomes play a critical in the clearance of macromolecules such as protein aggregates. The transcription factor TFEB is a master regulator of lysosomal function. TFEB responds to lysosomal stress and translocates to nucleus where it mediates gene expression. TFEB targets include subunits of the v-ATPase essential for lysosomal acidification. Here we investigated the role of TFEB-v-ATPase signaling in tau pathology.
METHOD: We created mutant mice in which the TFEB-v-ATPase signaling pathway is disrupted, and further crossed it with tau transgenic mice. We performed single nucleus RNA-sequencing (snRNA-seq) to examine the gene expression changes in various cell types. In addition, we performed immunostaining and biochemical analysis to characterize tau pathology and glial cell morphologies.
RESULT: We found that disruption of the TFEB-v-ATPase signaling leads to increased tau pathology, validating an important role of the lysosome in tau degradation. Surprisingly, we found that microglia can no longer be activated in the lysosomal mutants in response to tau pathology. snRNA-seq showed that microglia activation is associated with upregulation of both immune and lysosomal pathway genes. Reduced lysosomal TFEB-v-ATPase signaling leads to the downregulation of the immune pathways, demonstrating a critical role of the lysosome in microglia activation.
CONCLUSION: Our studies identify a novel lysosome-immune relationship in tauopathy and Alzheimer's disease.

DOI: https://doi.org/10.1002/alz70861_108383
Biochem J. 2025 Dec 23. pii: BCJ20253459. [Epub ahead of print]483(1):

Insights into PINK1/Parkin function and dysfunction from Drosophila models.

Seoyoung Park, Nikita Kozhushko, Thomas H Wight, Alexander J Whitworth.

  Loss-of-function mutations in PINK1 and PRKN cause familial forms of Parkinson's disease (PD). In vitro studies have revealed incredible insights into the molecular and cell-biological function of these genes, which have focused predominantly on mitophagy - the autophagic degradation of damaged mitochondria. The mechanisms of PINK1/Parkin function ultimately require investigation in an in vivo context using classic genetic approaches in animal models. In this context, Drosophila models have proven to be remarkably informative, in part due to robust phenotypes arising from null mutations. They have revealed important insights into the function of the Pink1 and parkin orthologues, much of which has proven to be conserved in humans. The simplicity, speed and genetic tractability make Drosophila an excellent in vivo model to interrogate the physiological functions of Pink1 and parkin and to rapidly test emerging hypotheses arising from in vitro work. They also represent a powerful model with which to explore the pathological consequences of Pink1/parkin loss in a whole-organism context. In this regard, several themes have emerged from recent studies that likely have significance for the neurodegenerative process in humans, including aberrant activation of immune signalling and consequent inflammation, disruptions to gut integrity and disturbed mitochondrial calcium handling. In this review, we evaluate the current evidence regarding the mechanism(s) of Pink1/parkin-mediated mitochondrial turnover in Drosophila, and discuss the potential implications of recent developments on the consequences of Pink1/parkin mutations and how these may inform the pathogenesis of PD.

Keywords:   Drosophila ; PINK1; Parkin; Parkinson’s disease; autophagy; calcium signalling; immune signalling; mitochondria; mitophagy; mtDNA; neurodegeneration

DOI:  https://doi.org/10.1042/BCJ20253459
Environ Pollut. 2025 Dec 19. pii: S0269-7491(25)01936-0. [Epub ahead of print] 127562

Trifloxystrobin induces oxidative stress-dependent activation of the OMA1-DELE1-HRI integrated stress response leading to apoptosis in human neuroblastoma cells.

Hanen Chaabani, Imen Ayed, Karima Rjiba, Salwa Abid, Joel Eyer, Damien Arnoult.

  Trifloxystrobin (TFX), a potent inhibitor of complex III in the mitochondrial respiratory chain, is a widely used strobilurin fungicide whose neurotoxic mechanisms remain poorly defined. This study investigated the molecular pathways underlying TFX-induced toxicity in human SH-SY5Y neuronal-like neuroblastoma cells, with particular emphasis on oxidative stress, mitochondrial dysfunction, and activation of the Integrated Stress Response (ISR). TFX exposure (24 h) exhibited an IC50 of approximately 100 μM, induced G0/G1 cell cycle arrest, and triggered mitochondria-mediated apoptosis, as evidenced by loss of mitochondrial membrane potential (ΔΨm), Bax activation, cytochrome c release, DNA fragmentation, phosphatidylserine exposure, and caspase-3 activation. These effects were accompanied by increased mitochondrial superoxide levels and decreased ATP production, indicating profound mitochondrial impairment. Pretreatment with N-acetylcysteine (NAC) markedly restored cell viability, reduced ROS accumulation, prevented ΔΨm dissipation, and diminished apoptotic damage. Mechanistically, TFX activated the ISR through the OMA1-DELE1-HRI mitochondrial stress signaling axis, as confirmed by loss-of-function experiments targeting these proteins. Importantly, both NAC and the ISR inhibitor ISRIB (Integrated Stress Response InhiBitor) significantly attenuated ISR activation and the resulting apoptosis, demonstrating that oxidative stress serves as an upstream trigger for ISR engagement and cell death. Collectively, these findings reveal that TFX induces oxidative stress-dependent activation of the OMA1-DELE1-HRI ISR pathway, linking mitochondrial dysfunction to apoptosis in human neuroblastoma cells. To our knowledge, this is the first report identifying ISR activation as a mechanistic component of strobilurin fungicide-induced neurotoxicity.

Keywords:  ISR; SH-SY5Y cells; Trifloxystrobin; apoptosis; oxidative stress

DOI:  https://doi.org/10.1016/j.envpol.2025.127562
Alzheimers Dement. 2025 Dec;21 Suppl 1 e098824

Basic Science and Pathogenesis.

Wesley H Brooks.

BACKGROUND: The "X chromosome-nucleolus nexus" hypothesis for lupus, describing mutual disruption of the inactive X and nucleoli, can assist in understanding Alzheimer's etiology. Since 49% of cellular pathways are affected in Alzheimer's, a systems approach is required, looking at multiple pathways and shared reactants like S-adenosylmethionine (SAM) and acetyl-CoA. Here, a new hypothesis is proposed for Alzheimer's describing mutual disruption of nucleoli and Alzheimer's related genes in peri-nucleolar chromatin.
METHOD: Key word searches (e.g., PubMed) retrieved relevant publications. Chromosome locations of Alzheimer's related genes and Alu element clusters were determined.
RESULT: The hypothesis "polyamine dysregulation and nucleolar disruption" emerged in which cellular stress can induce increased polyamine metabolism that wastes SAM, used in polyamine synthesis, and acetyl-CoA, used in polyamine recycling. Low SAM induces p38 kinase phosphorylation of Tau. Low acetyl-CoA leads to low acetylcholine seen in Alzheimer's. Polyamine changes drive nucleolar dynamics which disrupts peri-nucleolar chromatin. This can open clusters of Alu elements which can be expressed in abundance by RNA polymerase III. Alu RNA transcripts can bind nucleolin in competition with structural RNAs that normally bind nucleolin to stabilize the nucleolar shell. As the nucleolus loses its integrity, it becomes very inefficient, even fragmenting. In lupus this can release autoantigens, many of which contain nucleolar components. Alu elements comprise 11% of the genome but Alu clusters exist, for example, in chromosome 19 with the ApoE4 allele, Alu elements comprise 25.8%. Nucleolar disruption can disrupt epigenetic control of Alzheimer's related genes in peri-nucleolar chromatin, such as PSEN1 on chromosome 14 and APP on chromosome 21. With the appearance of hyperphosphorylated Tau, there can be aggregation by increased polyamines.
CONCLUSION: The "polyamine dysregulation and nucleolar disruption" hypothesis describes how stress can lead to extraordinary nucleolar dynamics, disrupting epigenetic control of Alzheimer's related genes. In addition, Alu clusters reside in peri-nucleolar chromatin with the possibility that significant Alu expression could further disrupt the nucleoli. Also, there are sequestered polyamine metabolism alleles located in the inactive X that could become active. Wasteful polyamine metabolism reduces SAM and acetyl-CoA leading to Tau hyperphosphorylation by p38 kinase and low acetylcholine seen in Alzheimer's.

DOI: https://doi.org/10.1002/alz70855_098824
Alzheimers Dement. 2025 Dec;21 Suppl 1 e097357

Basic Science and Pathogenesis.

Mariana Chauvet, Danielle Cozachenco Ferreira, Alinny Isaac, Sergio T Ferreira, Mychael V Lourenco.

BACKGROUND: Alzheimer's disease (AD) is the leading cause of dementia in elderly humans worldwide. Brain mRNA translation (protein synthesis) is essential for synaptic plasticity and cognition, and converging evidence indicates it is impaired in AD. Protein synthesis is composed of three major steps: initiation, elongation and termination. There are several factors involved in all three steps that are essential to ensure proper translation control. In particular, the key factors of the initiation and elongation steps are the eukaryotic initiation factor 2 alpha (eIF2a) and eukaryotic elongation factor 2 (eEF2), respectively, whose aberrant phosphorylation indicates translational repression. Importantly, failures in translational control are associated with several pathological conditions, including AD. Our group and others have shown that global protein synthesis is attenuated in AD neurons. However, the precise roles of eIF2a and eEF2 (and their phosphorylation status) in AD and other causes of dementia are not yet fully understood.
METHOD: Phosphorylated and total eIF2a and eEF2 were measured by Western blotting in postmortem hippocampi and prefrontal cortices from healthy controls (HC) and AD patients of a Brazilian cohort.
RESULT: We found that phospho-eIF2a and phospho-eEF2 levels are increased in individuals with higher CERAD amyloid score in the hippocampus and prefrontal cortex. Similarly, both phospho-eIF2a and phospho-eEF2 levels are increased in individuals with higher Braak stages in the prefrontal cortex.
CONCLUSION: Together, these results suggest that these translational factors may be differently altered in relation to amyloid and tau pathology in AD.

DOI: https://doi.org/10.1002/alz70855_097357
bioRxiv. 2025 Dec 16. pii: 2025.12.14.694244. [Epub ahead of print]

Intracellular aggregation of the transthyretin protein is limited by Hsp70 chaperones.

Claire M Radtke, Adam S Knier, Sean A Martin, Jane E Dorweiler, Matt E Hudson, Anita L Manogaran.

Extracellular amyloid deposits are a hallmark feature of systemic protein aggregation diseases such as transthyretin amyloidosis (ATTR). However, emerging evidence suggests that extracellular transthyretin (TTR) aggregates are internalized and result in an intracellular stress response, including elevated Hsp70 levels. While ATTR research has predominantly focused on extracellular TTR amyloid, our understanding of TTR aggregation inside the cell is poorly explored. To better understand how intracellular chaperones impact intracellular TTR, we used a yeast model that expresses TTR fused eGFP (TTR-eGFP) within the cytoplasm. Since the Hsp70 chaperone family, and co-chaperones the J-domain proteins (JDPs) and Hsp110, act as a disaggregase in vitro, we asked how these molecular chaperones impact TTR aggregation intracellularly in vivo . TTR-eGFP forms detergent-soluble high molecular weight (HMW) aggregates in yeast that have biochemistry profiles similar to human patient TTR. While knockdown of the JDP, Sis1, and deletion of the Hsp110, Sse1, appear to slightly increase TTR-eGFP aggregation, the loss of two major yeast Hsp70s, Ssa1 and Ssa2, lead to a significant increase in the size of HMW species. Taken together, our data suggest that Hsp70s limit the formation of HMW TTR aggregates in the intracellular environment. Based on our results, it is possible that the age-related decline of protein homeostasis, including Hsp70s, may promote the intracellular aggregation of TTR.

DOI: https://doi.org/10.64898/2025.12.14.694244
Nucleic Acids Res. 2025 Nov 26. pii: gkaf1311. [Epub ahead of print]53(22):

Suboptimal codon pairs trigger ribosome collisions and cellular quality control responses in tRNA modification mutants.

Jie Wu, Cristian Eggers, Olga Sin, Łukasz Koziej, Hector Mancilla, Fabienne Mollet, Hans R Schöler, Hannes C A Drexler, Tristan Ranff, Christian Fufezan, Claudine Kraft, Sebastian Glatt, Jan M Bruder, Sebastian A Leidel.

Transfer RNA (tRNA) modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding. However, the mechanisms by which tRNA modifications modulate codon optimality and trigger phenotypes remain unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs in wobble uridine modification (U34) mutants. This triggers ribosome collisions and a coordinated hierarchical response of cellular quality control pathways. High-resolution ribosome profiling reveals an unexpected functional diversity of U34 modifications during decoding. For instance, 5-carbamoylmethyluridine (ncm5U) exhibits distinct effects at the A and P sites. Importantly, ribosomes only slow down at a fraction of codons decoded by hypomodified tRNA, and the decoding speed of most codons remains unaffected. However, the translation speed of a codon largely depends on the identity of A- and P-site codons. Stalling at modification-dependent codon pairs induces ribosome collisions, triggering ribosome-associated quality control (RQC) and preventing protein aggregation by degrading aberrant nascent peptides and messenger RNAs. Inactivation of RQC stimulates the expression of molecular chaperones that remove protein aggregates. Our results demonstrate that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs, showing the coordinated regulation and adaptability of cellular surveillance systems. These systems ensure efficient and accurate protein synthesis and maintain protein homeostasis.

DOI: https://doi.org/10.1093/nar/gkaf1311
Protein Sci. 2026 Jan;35(1): e70428

Defining substrate specificities of human RNA capping methyltransferases through quantitative assessment of independent yet cooperative activities.

Fatemeh Taherian, Mark F Mabanglo, Taraneh Hajian, Sarah Tucker, Emilija Kalinic, Sumera Perveen, Ahmed Aman, Masoud Vedadi.

  Human RNA capping is critical for mRNA splicing, protection of RNA from 5' exonucleases in the cytoplasm, and targeting to the ribosome. Human RNMT, CMTR1, and CMTR2 are RNA methyltransferases involved in the RNA capping process. They play a significant role in the proliferation and differentiation of embryonic stem cells and have been implicated in cancer. Substrate specificities of human RNA capping methyltransferases have been somewhat explored in a few studies. Here, we report on a comprehensive, systematic, and quantitative assessment of their substrate specificities along with SARS-CoV-2 counterparts, nsp14 and nsp16. We discovered novel cooperative activities of human enzymes. We designed and synthesized various RNA substrates with defined patterns of methylation to systematically assess the dependency or cooperativity of their activities using radiometric assays followed by mass spectrometry to verify RNA methylation status. We have tested all five enzymes in parallel against these substrates and determined kinetic parameters. Our data not only indicate that the catalytic activities of human RNMT, CMTR1, and CMTR2 are distinct and nonoverlapping, but also provide a novel quantitative assessment of their activities, indicating how and to what extent these proteins affect each other's function. Unlike nsp14 and nsp16, their functions are not necessarily sequential, but show significant cooperativity. Altogether, our data provide a comprehensive understanding of substrate specificities of human RNA capping methyltransferases, enabling the development of potential future anticancer therapeutics and assessment of antiviral therapeutics' selectivity.

Keywords:  CMTR1; CMTR2; RNA capping; RNA methylation; RNMT

DOI:  https://doi.org/10.1002/pro.70428
bioRxiv. 2025 Dec 09. pii: 2025.12.09.693200. [Epub ahead of print]

Uncovering the regulatory network of the small RNA SuhB and its contribution to stress resistance in Sphingopyxis granuli TFA.

Inmaculada García-Romero, Alberto Pires-Acosta, Belén Floriano, Francisca Reyes-Ramírez.

Post-transcriptional regulation by small RNAs (sRNAs) enables bacteria to fine-tune gene expression and rapidly adapt to fluctuating environmental conditions. In Sphingopyxis granuli TFA, SuhB, the only sRNA characterized to date in this strain, was previously shown to repress thnR translation to control tetralin degradation under carbon catabolite repression conditions. Here, we reveal additional regulatory roles of SuhB beyond carbon metabolism. Deletion of suhB increases sensitivity to diverse abiotic stresses, including osmotic, oxidative, desiccation, and copper stress. Label-free quantitative proteomic analysis indicates widespread alterations in the proteome in the absence of SuhB, affecting metabolic pathways and membrane-associated processes. Moreover, a LysR-type transcription factor mutant, identified as a direct activator of suhB, shows similar phenotypes. Together, these findings demonstrate that SuhB functions as a global post-transcriptional regulator, coordinating metabolic balance, membrane composition, and stress resistance in S. granuli TFA, highlighting the critical role of sRNA-mediated regulation in environmental bacteria.

DOI: https://doi.org/10.64898/2025.12.09.693200
J Neuromuscul Dis. 2025 Dec 23. 22143602251409302

Identification of myokines associated with the pathological stress response in the mdx mouse model of Duchenne muscular dystrophy.

Erynn E Johnson, Jacob Powers, James M Ervasti.

   PURPOSE: Skeletal muscle constitutes 30-40% of total body mass and is now considered an endocrine organ, given its secretion of a variety proteins, metabolites, and cytokines. We have previously shown that the absence of dystrophin in skeletal muscle contributes to lethal systemic stress pathology in the mdx mouse model of Duchenne muscular dystrophy through a mechanism that remains to be identified. Here we searched for secreted protein signaling factors, or myokines, released from dystrophin-deficient skeletal muscle that influence the organism-wide integrated stress response. We performed skeletal muscle extracellular fluid extraction and discovery proteomics for wild-type, mdx, and transgenic mdx mice rescued by expression of a dystrophin construct, all analyzed under basal conditions and following brief scruff restraint stress that causes inactivity in mdx mice.
MAJOR FINDINGS: Our analysis demonstrated that skeletal muscle dystrophinopathy is associated with increased expression of numerous proteins in both intact mdx skeletal muscle and extracellular fluid compared to healthy mice. Brief scruff restraint revealed protein candidates with differential abundance in mdx extracellular fluid. Specifically, altered follistatin-like 1 protein and adiponectin secretion in response to scruff stress was shown to be dependent on skeletal muscle dystrophinopathy. The diverse signaling roles of follistatin-like 1 in the cardiovascular, musculoskeletal, and nervous system implicate it as a particularly intriguing myokine candidate regulating the mdx stress response.
CONCLUSIONS: Our current study informs on the skeletal muscle secretory profile in mdx following a stressful stimulus and provides new leads to elucidate the mechanism by which mdx skeletal muscle orchestrates inter-organ stress signaling.

Keywords:  duchenne muscular dystrophy; myokine; proteomics; skeletal muscle; stress physiology

DOI:  https://doi.org/10.1177/22143602251409302
J Vis Exp. 2025 Dec 05.

Estimation of Telomeric Repeat-containing RNA from DNA/RNA Hybrid Complexes.

Ecmel Mehmetbeyoglu Duman, Zeynep Yilmaz Sukranli, Hayriye Kilci, Serpil Taheri, Minoo Rassoulzadegan.

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from all telomeres in higher eukaryotes, and only a subset of these molecules forms stable DNA/RNA hybrids at telomeres. We have outlined a detailed molecular protocol to identify and purify these hybrid estimates. This streamlined method enables the direct extraction of DNA/RNA hybrid interactions naturally formed in vivo and can be applied to detect all such regions. By minimizing procedural steps, this technique is highly efficient in isolating RNA attached to DNA, estimating the reliability and reproducibility of downstream sequencing. Guanidine isothiocyanate reagent is used for total cell lysis and homogenization, preserving the integrity of nucleic acids (DNA and RNA), particularly DNA/RNA hybrids. Adding chloroform initiates phase separation via centrifugation (three distinct layers): a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA, released from both the nucleus and cytoplasm, is retained in the aqueous phase, while high-molecular-weight DNA, including the DNA/RNA hybrid regions, is confined to the interphase. With isopropyl alcohol, followed by centrifugation, the aqueous phase (free RNA), interphase, and DNA, particularly DNA/RNA hybrids, are recovered. To remove the remaining traces of proteins, the sample is incubated with Proteinase K. Treatment with DNase, phenol/chloroform extraction, isopropyl alcohol, and centrifugation is used to release RNA hybridized to DNA.

DOI: https://doi.org/10.3791/67984
Mar Drugs. 2025 Dec 02. pii: 464. [Epub ahead of print]23(12):

Mechanistic Investigation of Adociaquinone and Xestoquinone Derivatives in Breast Cancer Cells.

Yu-Dong Zhou, Fakhri Mahdi, Nicholas M Nagle, Mika B Jekabsons, Dale G Nagle.

  Xestoquinone derivatives isolated from marine sponges exhibit a range of bioactivities, including the inhibition of HIF signaling, mitochondrial function, and tumor cell proliferation. Mechanistic investigation suggested that 14-hydroxymethylxestoquinone (1) acts as a protonophore. Although adociaquinones A (5) and B (6) each stimulated cellular oxygen consumption, neither affected mitochondrial membrane potential. Cell-based respiration studies revealed that adociaquinones restored sodium azide-stalled oxygen consumption and ascorbate enhanced this response, suggesting ascorbate-supported redox cycling as a possible mechanism by which adociaquinones suppress HIF and tumor cell proliferation. These xestoquinone derivatives activated cellular stress response pathways that inhibit protein translation by phosphorylating key regulatory proteins (i.e., eIF2α, eIF4E, and eEF2). Further, thiol-reducing agents NAC and DTT attenuated the monosubstituted xestoquinone derivatives' efficacy to inhibit HIF signaling, suggesting a potential mechanism of action that involves sulfhydryl modification.

Keywords:  HIF-1; adociaquinone; breast cancer cells; mechanism of action; redox cycling; respiration; stress response; xestoquinone

DOI:  https://doi.org/10.3390/md23120464
Nucleic Acids Res. 2025 Nov 26. pii: gkaf1386. [Epub ahead of print]53(22):

An RBPMS-driven splicing regulatory axis including MBNL1, RBFOX2, and QK promotes smooth muscle cell contractile identity.

Yuling Huang, Rafael Kollyfas, Ruth Partridge, Clare Gooding, Sanjay Sinha, Irina Mohorianu, Aishwarya Jacob, Christopher W J Smith.

Phenotype switching of vascular smooth muscle cells (SMCs) between contractile and more proliferative and motile states is associated with cardiovascular disease and is underpinned by transcriptional and alternative splicing (AS) programs. We previously showed the RNA-binding protein (RBP) RNA Binding Protein with Multiple Splicing (RBPMS) to be a master regulator of AS in differentiated SMCs. Although changes in master regulator activities can drive AS programs, such proteins rarely act alone. Here we investigated how MBNL1, RBFOX2, and QK act as coregulators with RBPMS to promote contractile smooth muscle AS and phenotypic properties. All four RBPs largely promoted contractile phenotype splicing, with RBPMS showing the highest degree of alignment with the program. Coregulated splicing events were enriched for functions associated with actin filaments and focal adhesions indicating RBPMS-coordinated remodelling of the cellular contractile and motility machinery. Strikingly, while knockdown of each RBP affected various cell morphological and functional properties, knockdown of RBPMS alone induced all aspects of phenotype switching, including lower contraction, higher proliferation, and motility. Our results highlight how a master regulatory RBP can guide an axis of more widely expressed regulators to drive key cellular phenotype changes independently of a transcriptional program.

DOI: https://doi.org/10.1093/nar/gkaf1386
Nat Commun. 2025 Dec 22. 16(1): 11340

Vaccinia virus mRNAs containing long 5'-poly(A)-leaders lack a canonical 5'-methylguanosine cap.

Václav Vopálenský, Michal Sýkora, Zora Mělková, Ivan Barvík, Kamila Horáčková, Tomáš Mašek, Martin Pospíšek.

The vaccinia virus (VACV) is a prototypical poxvirus that was originally used to eradicate smallpox. Half a century ago, investigation into VACV mRNA substantially contributed to the fundamental discovery of the 5' mRNA cap, a hallmark of all eukaryotic and many viral mRNAs. VACV research also facilitated the identification and understanding of the general mechanism of 5' mRNA cap synthesis. We analyzed VACV transcripts at the level of individual mRNA molecules using a modified 5' RACE method. Our results demonstrate that VACV mRNAs containing long nontemplated 5' poly(A) leaders lack the 5' cap structure in vivo. The probability of the m7G cap occurrence decreases with the increasing number of nontemplated adenosines in the 5' poly(A) leader. Although half of VACV mRNAs with a single nontemplated adenosine still contain the m7G cap, only about 4% of viral mRNAs with leaders consisting of six or more nontemplated adenosines retain the cap. Uncapped mRNA can be transcribed from all genes containing adenosine-rich initiator sequences (INR) within their promoters. Early genes with INR still produce mostly capped transcripts (40%-59%, depending on the gene). However, intermediate mRNAs are less capped (11%-56%) and late mRNAs are mostly uncapped (0%-10% of capped mRNAs, depending on the gene).

DOI: https://doi.org/10.1038/s41467-025-67916-w
PLoS Biol. 2024 Apr;22(4): e3002603

Multiple checkpoints ensure ribosomes have the correct end.

Jacob Gordon, Robin E Stanley.

The 3' end of the 18S ribosomal RNA is formed by the endoribonuclease Nob1. How cells ensure the accuracy of the 3' end has remained a mystery. A new study in PLOS Biology revealed that there are multiple checkpoints to ensure that only ribosomes containing the correct 3' end participate in translation.

DOI: https://doi.org/10.1371/journal.pbio.3002603
Proteomes. 2025 Nov 25. pii: 63. [Epub ahead of print]13(4):

Azidohomoalanine (AHA) Metabolic Labeling Reveals Unique Proteomic Insights into Protein Synthesis and Degradation in Response to Bortezomib Treatment.

Lina Alhourani, Yasser Tabana, Ashwin Anand, Richard P Fahlman.

   BACKGROUND: Multiple myeloma (MM) is essentially an incurable cancer, but treatments with proteasome inhibitors are widely used clinically to extend patient survival. While the mechanisms of proteasome inhibition by Bortezomib are well known, the cellular responses to this proteotoxic stress that leads to sensitivity by MM are not fully elucidated. This study reports on the application of an emerging method to investigate proteostasis by proteomics.
METHODS: We utilized metabolic labeling with azidohomoalanine (AHA) in a MM cell line in combination with Bortezomib treatment. AHA labeling facilitates the selective isolation and identification of proteins for investigations of protein synthesis or protein degradation.
RESULTS: The data collected reveals significant changes in gene protein synthesis upon Bortezomib treatment, including protein neddylation. The data also reveals a global increase in protein degradation, which suggests the induction of an autophagy-related process. The resulting data collected reveals significant changes upon Bortezomib treatment in protein synthesis of genes, including protein neddylation, and protein degradation data reveals a global increase in protein degradation, suggesting an induction of an autophagy-related process. Subsequent cellular and proteomic analysis investigated the additional treatment of an autophagy inhibitor, hydroxychloroquine, in combination with Bortezomib treatment by label-free proteomics to further characterize the proteome-wide changes in these two proteotoxic stresses.
CONCLUSIONS: AHA metabolic labeling proteomics to investigate protein synthesis and degradation enables novel complementary insights into complex cellular responses compared to that of traditional label-free proteomics.

Keywords:  azidohomoalanine; proteasome; protein degradation; protein synthesis; proteomics; proteostasis

DOI:  https://doi.org/10.3390/proteomes13040063
Front Cell Dev Biol. 2025 ;13 1731227

Emerging roles of stress granules in liver diseases: a comprehensive review.

Qianyun Zhang, Yun Liu, Junzhe Tang, Ming Li, Qiang Jia.

  Stress granules (SGs) are transient, membraneless condensates that assemble dynamically within cells in response to diverse stressors. In recent years, SGs have been found to be closely associated with multiple pathological states and have attracted significant attention, particularly concerning their roles in hepatic pathophysiology. Functioning as critical hubs for post-transcriptional regulation, SGs maintain cellular homeostasis through the sequestration, transport, and translational suppression of mRNA, thereby potentially modulating the initiation and progression of various liver diseases. Our review summarizes the assembly mechanisms of SGs and recent research advances concerning their involvement in diseases including hepatocellular carcinoma, viral hepatitis, acute liver injury and fatty liver disease. It particularly focuses on SGs core RNA-binding proteins and associated regulatory networks. Although research into the impact of SGs on liver diseases remains in a nascent phase, with mechanistic details still elusive, SGs emerge as pivotal molecular nexuses connecting cellular stress responses to pathophysiological states, highlighting their therapeutic potential for liver disorders. This review aims to provide a theoretical foundation for a deeper understanding of the roles of SGs in liver pathologies and to promote their further development in both fundamental research and clinical translation.

Keywords:  acute liver injury; fatty liver disease; hepatocellular carcinoma; stress granules; viral hepatitis

DOI:  https://doi.org/10.3389/fcell.2025.1731227
Emerg Top Life Sci. 2025 Dec 24. pii: ETLS20253009. [Epub ahead of print]9(6):

Five hundred million years of hunger: how animals evolved to survive essential amino acid scarcity.

Benjamin S Pickard.

  Proteins are the machinery for the processes of life. Each protein is made up of a defined combination of 20 building blocks, the amino acids. The animal kingdom is distinguished from most other forms of life by a half-billion-year-old choice to relinquish the synthesis of 9 of the 20 amino acids and instead rely on their dietary acquisition for protein synthesis. From that point onwards, animals entered into a permanent and obligatory hunt for these 'essential amino Acids' (EAAs). This perspective states that this seemingly destructive event was, in fact, foundational for the animal kingdom. Hypotheses for its origins are discussed, including a newly observed bias in EAA codon nucleotide composition that may help economise their use in proteins during scarcity. Tight restrictions on the inclusion of EAAs in protein sequences would be expected, but a minority of proteins with extreme EAA compositions are found. It is hypothesised that such proteins act as sentinels of EAA shortage in the diet, prompting beneficial responses from the organism. The control of hunger behaviours and reproductive timing are two processes in which EAA-rich proteins may be important. The leptin pathway of hunger behaviour regulation and reproductive development, traditionally associated with bodily lipid homeostasis, may be sensitive to EAA levels through this sequence-based mechanism. EAAs appear to have been a strong force in animal evolution. The biology emerging from their patterns of use in our proteins provides a direct link between nutritional state and specific biological processes - a coherent route to better dietary interventions in the future.

Keywords:  amino acid metabolism; evolutionary biology; gene expression and regulation; homeostasis; obesity; protein synthesis

DOI:  https://doi.org/10.1042/ETLS20253009
bioRxiv. 2025 Dec 18. pii: 2025.12.17.694926. [Epub ahead of print]

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

Daniel J Foust, Frances C Lowder, Jessica Chung, Julianna R Cresti, Lieke A van Gijtenbeek, Lyle A Simmons, Julie S Biteen.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R-loops during transcription. R-loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis : DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photactivated localization microscopy. In this work, we find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo .
Significance: RNA-DNA hybrids (RDHs) are essential, transient intermediates in DNA replication, yet their presence significantly increases the susceptibility of the genome to damage. We characterized the single-molecule behavior of three proteins important for processing RDHs in Okazaki fragments in living bacteria. We find that enzyme activity is modulated by access to the replisome and nucleoid. Specifically, we find that DNA polymerase I is preferentially localized to the replisome, while FenA and RNase HIII dwell times at the replisome are very short and below our detection limit. Our work shows that Pol I, FenA, and RNase HIII turn over rapidly in cells, providing new insight into how lagging-strand replication is coordinated in vivo .

DOI: https://doi.org/10.64898/2025.12.17.694926
bioRxiv. 2025 Dec 16. pii: 2025.12.12.693499. [Epub ahead of print]

Molecular profiling of glioblastoma-derived extracellular vesicles identifies small nucleolar RNAs as candidate liquid biomarkers for radiation-induced senescence.

Valerie DeLuca, Nathaniel Hansen, Priya Digumarti, Nanyun Tang, Karen Fink, George Snipes, Patrick Pirrotte, Michael Berens.

Radiation-induced senescence (RIS) in glioblastoma (GBM) is an undesirable cell fate that inhibits tumor cell death and supports resistance and outgrowth. While senescence-targeting drugs are promising adjuvants, their clinical application will require proper patient selection based on post-treatment RIS burden. Current methods to evaluate senescence, however, are tissue-based, and given GBM's difficult anatomical location, post-treatment biopsies are impractical. Therefore, novel and less invasive biomarkers for TIS are urgently needed. To this end, we aimed to identify candidate extracellular vesicle (EV) liquid biomarkers for TIS by profiling senescence-associated cargo changes within GBM EVs. Using a panel of GBM patient-derived cell lines, we show that RIS is the primary functional state following radiation exposure and is associated with significant alterations in the cargo of senescent-derived EVs (senEVs). In particular, senEV transcriptomes have an increased abundance of senescence-associated RNA species and enrichment of senescence-associated gene sets. Most striking, however, was that senEVs are most differentiated by the significant enrichment of a panel of snoRNAs. This signature was conserved in 4/5 GBM models of RIS and was validated by qRT-PCR. Further analysis by mass spectrometry revealed that snoRNAs are likely co-packaged with their associating proteins, as senEVs had concurrent increases in these binding partners. Finally, in a preliminary patient cohort comparing plasma EVs obtained prior to surgery to those obtained after completion of their radiation therapy, we identified increased senescence-associated RNA such as CDKN2B and GLB1 and the snoRNA SNORA49 in post-radiation EVs. Altogether, this data suggests that senEV RNA species, and particularly snoRNAs, are a promising analyte for RIS-biomarker development. With further study, this work may open avenues for a companion diagnostic for senotherapeutics.

DOI: https://doi.org/10.64898/2025.12.12.693499
Protein Sci. 2026 Jan;35(1): e70388

Purification and characterization of recombinant human translation initiation factor eIF3.

Irene Diaz-Lopez, Yuliya Gordiyenko, Philipp K Zuber, Xueyan Li, V Ramakrishnan.

  Eukaryotic translation initiation factor 3 (eIF3) is an essential factor in protein synthesis. In mammals, it is a ~800 kDa complex composed of 12-13 subunits. Biochemical and mechanistic studies of the function of eIF3 and its individual subunits in translation require purified eIF3. However, current strategies for obtaining mammalian eIF3 rely on purification of the endogenous factor from cultured human cells or rabbit reticulocytes lysates, both of which are expensive and time consuming. Here we present a recombinant insect-cell expression and purification system for human eIF3 which allows the purification of large amounts of functional, homogeneous eIF3 efficiently and cost-effective, while also enabling engineering, such as generation of site-specific mutations in the factor for functional studies.

Keywords:  eIF3; high yield; multifactor complex purification; translation initiation

DOI:  https://doi.org/10.1002/pro.70388
Nucleic Acids Res. 2025 Nov 26. pii: gkaf1368. [Epub ahead of print]53(22):

Pseudouridine increases ribosome stability in a thermophilic eukaryote.

Klemens Wild, Marius Klein, Alicia Burkard, Virginie Marchand, Nikola Kellner, Antonio Paez, Stefan Pastore, Tamer Butto, Yuri Motorin, Mark Helm, Irmgard Sinning.

RNA modifications alter stability, folding space, and interaction network of RNA molecules. Ribosomal RNA (rRNA) modifications stabilize the structure of ribosomes and cluster around functionally important sites such as the peptidyl transferase center, ribosomal subunit bridges, and the polypeptide tunnel. Here, we investigate the rRNA modifications of the thermophilic fungus Chaetomium thermophilum (ct), a model organism for eukaryotic thermophily and structural stability. Using LC-MS/MS, orthogonal second and third generation RNA-sequencing and high-resolution cryo-electron microscopy, we describe a cross-correlating method to assign and quantify all ct rRNA modifications. Overall, a doubling of rRNA modifications to 4% explains ribosomal thermostability with an extended distribution towards peripheral functional sites. The 2.4 Å structure of the idle ct60S ribosome, retaining nascent chains and including metal ions, polyamines, and water molecules, allows for a comprehensive structure-function analysis. Comparison with mesophilic ribosomes from Chaetomium globosum, yeast, and human highlights the significant increase of pseudouridines (Ψs). The number of Ψs linearly correlates with growth temperature, suggesting statistical modification. A ct-specific Ψ substitution forming a 'Ψ-turn' at the polypeptide tunnel exit close to the third constriction exemplifies mechanistic adaptations of the ribosome at elevated temperatures.

DOI: https://doi.org/10.1093/nar/gkaf1368