Biology subjects

Allen, G. E.

Publications and source records attributed to Allen, G. E..

9 recordsLinked to original sources

bioRxiv2026

Molecular factors shaping small RNA content in mouse sperm

In mammalian reproduction, sperm and oocytes fuse to generate embryos. In contrast to maternally inherited small RNAs, sperm-borne small RNAs have been implicated in embryo viability but remain incompletely understood. Unanswered questions about molecular forces shaping small RNA content in sperm and sources of reporting discrepancies hinder a deeper understanding of their post-fertilization roles. Here, we investigate the dynamics and protein association of small RNAs in C57BL/6 mice during spermiogenesis and sperm capacitation. Using unique molecular identifiers and spike-ins to minimize technical biases in low-input sequencing, we demonstrate a selective sperm small RNA repertoire derived from a subset of spermatogenic small RNAs that remains malleable during capacitation. Retained PIWI-interacting RNAs persist despite widespread RNA decay and are insensitive to capacitation, which may be partly attributed to their association with MIWI proteins. Our data reveal RNA species susceptible to PCR duplicate-related abundance overestimation and uncover biological and technical factors shaping experimentally observed sperm small RNA profiles. These findings refine our understanding of sperm-borne RNAs and inform future functional studies and medically assisted reproduction.

molecular biology↗

bioRxiv2026

Intrinsic features of the RNase E membrane targeting sequence specify RNA degradosome organisation and activity

In bacteria, transcription and RNA degradation are physically separated via segregation of the main ribonucleolytic machinery - the RNA degradosome - into phase-separated or membrane-anchored molecular assemblies driven by RNase E. Despite the widespread conservation of an amphipathic membrane anchor (MTS) in RNase E, the regulatory information embedded within this sequence and its biological importance remain poorly understood. Here, we have studied the importance of the Pseudomonas aeruginosa RNase E MTS for bacterial fitness or virulence and assessed its interchangeability. We show that amphipathicity is dispensable for foci scaffolding but necessary for proper foci morphology, dynamics, and localisation, although sequence modulates foci behaviour. Loss of the MTS additionally causes a drastic sensitivity to high salinity and a consistent virulence defect in Galleria mellonella larvae. Moreover, transcriptomics and analysis of mRNA spatial organisation reveal that the MTS mutant has specific stabilisation of localised membrane protein-encoding transcripts, together with abnormal operon processing. Altogether, our study highlights the elegant MTS-mediated control of spatial organisation and target selection, shaping the transcriptome and bacterial stress response.

microbiology↗

bioRxiv2026

Puf3 contributes to changes in mRNA solubility, translation elongation dynamics at rare arginine codons and loss of protein homeostasis in cells lacking Not4

The Not proteins of the Ccr4-Not complex regulate translation elongation dynamics, essential for proper folding and assembly of new proteins. In yeast, ribosomes with non-optimal codons in the A-site are enriched within the pool of ribosomes bound by Not4 and Not5. Such ribosomes accumulate in cells lacking Not4 or Not5 that show defects in co-translational assembly and aggregation of new proteins. Recently we observed that depletion of Not1 and Not4 inversely regulate changes in mRNA solubility, correlating with inverse codon-specific changes in A-site ribosome dwelling occupancies (RDOs). Here we describe that mRNAs less soluble upon Not4 depletion are enriched for targets of the RNA-binding protein Puf3. We determine that Puf3 contributes to inverse changes of A-site RDOs upon Not1 and Not4 depletion, in particular at rare arginine codons, and it contributes to changes in mRNA solubility in not4{Delta}. Moreover, deletion of Puf3 suppresses temperature sensitivity and protein aggregation in the not4{Delta} strain, while overexpression of Puf3 is toxic. Puf3 post-translational modifications and the Puf3 interactome are altered in not4{Delta}. Taken together, our results associate alterations in Puf3 post-translational status and function, including contribution to translation elongation dynamics, with not4{Delta} mutant phenotypes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/695943v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@855afdorg.highwire.dtl.DTLVardef@118addaorg.highwire.dtl.DTLVardef@13dd015org.highwire.dtl.DTLVardef@12824c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Critical functions and key interactions mediated by the RNase E scaffolding domain in Pseudomonas aeruginosa

The RNA degradosome is a bacterial multi-protein complex mediating mRNA processing and degradation. In Pseudomonadota, this complex assembles on the C-terminal domain (CTD) of RNase E through short linear motifs (SLiMs) that determine its composition and functionality. In the human pathogen Pseudomonas aeruginosa, the RNase E CTD exhibits limited similarity to that of model organisms, impeding our understanding of RNA metabolic processes in this bacterium. Our study systematically maps the interactions mediated by the P. aeruginosa RNase E CTD and highlights its critical role in transcript regulation and cellular functions. We identified the SLiMs crucial for membrane attachment, RNA binding and complex clustering, as well as for direct binding to the core components PNPase and RhlB. Transcriptome analyses of RNase E CTD mutants revealed altered expression of genes involved in quorum sensing, type III secretion, and amino acid metabolism. Additionally, we show that the mutants are impaired in cold adaptation, pH response, and virulence in an infection model. Overall, this work establishes the essential role of the RNA degradosome in driving bacterial adaptability and pathogenicity. Author summaryBacteria must rapidly adapt to changing environments, whether facing temperature shifts, nutrient scarcity, or antibiotic exposure. A key mechanism enabling this adaptability is the regulation of mRNA levels--the molecular blueprints for protein production. This process is governed by the RNA degradosome, a multi-protein complex that processes and degrades RNA to control gene expression. Although the RNA degradosome core function is conserved across bacteria, its composition and organization differ significantly between species, reflecting diverse lifestyles and environmental challenges each bacterium encounter. In this study, we investigated the RNA degradosome in Pseudomonas aeruginosa, a bacterial pathogen causing difficult-to-treat infections in humans. We identified its components and mapped regions within the complex essential for RNA binding, membrane attachment, and spatial organization. Disrupting these regions compromised P. aeruginosa ability to survive in cold conditions, respond to stress, and establish an infection. Through this work, we uncovered unique features of the P. aeruginosa RNA degradosome that distinguish it from those of other species, emphasizing the RNA degradosome critical role in bacterial adaptability and highlight it as a promising target for therapies against P. aeruginosa infections.

microbiology↗

bioRxiv2024

Cytosolic N6AMT1-dependent translation supports mitochondrial RNA processing

Mitochondrial biogenesis relies on both the nuclear and mitochondrial genomes, and imbalance in their expression can lead to inborn error of metabolism, inflammation, and aging. Here, we investigate N6AMT1, a nucleo-cytosolic methyltransferase that exhibits genetic co-dependency with mitochondria. We determine transcriptional and translational profiles of N6AMT1 and report that it is required for the cytosolic translation of TRMT10C (MRPP1) and PRORP (MRPP3), two subunits of the mitochondrial RNAse P enzyme. In the absence of N6AMT1, or when its catalytic activity is abolished, RNA processing within mitochondria is impaired, leading to the accumulation of unprocessed and double-stranded RNA, thus preventing mitochondrial protein synthesis and oxidative phosphorylation. Our work sheds light on the function of N6AMT1 in protein synthesis and highlights a cytosolic program required for proper mitochondrial biogenesis.

molecular biology↗

bioRxiv2024

Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria

RNA helicases--central enzymes in RNA metabolism-- often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

molecular biology↗

bioRxiv2024

Reprogramming neuroblastoma by diet-enhanced polyamine depletion

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/573662v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1fdbaecorg.highwire.dtl.DTLVardef@fb03f4org.highwire.dtl.DTLVardef@1c0afa7org.highwire.dtl.DTLVardef@14c384f_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Extra-tumoral conversion of arginine feeds tumor ornithine via uptake from circulation in MYCN-neuroblastoma. - A proline and arginine free diet enhances pharmacological polyamine depletion via reduced ornithine substrate availability. - Polyamine depletion disrupts oncogenic translation to induce a pro-differentiation proteome causing neuroblast differentiation and prolonged survival in the TH-MYCN mouse model. - Genes of specific cellular programs have evolved codon usage preferences that enable coherent translational rewiring in response to metabolic stress, such as polyamine depletion.

cancer biology↗

bioRxiv2021

eIF4E3 Forms an Active eIF4F Complex during Stresses (eIF4FS) Targeting mTOR and Re-Programs the Translatome.

The eIF4E are a family of initiation factors that bind the mRNA 5 cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active only during stress and that it selects specific mRNA populations based on 5 TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have "moon-lighting" functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote "stress resistance" and adaptation.

molecular biology↗

bioRxiv2019

Switch from translation initiation to elongation needs Not4 and Not5 collaboration

Not4 and Not5 are crucial components of the Ccr4-Not complex with pivotal functions in mRNA metabolism. Both associate with ribosomes but mechanistic insights on their function remain elusive. Here we determine that Not5 and Not4 synchronously impact translation initiation and Not5 alone alters translation elongation. Deletion of Not5 causes elongation defects in a codon-dependent fashion, increasing and decreasing the ribosome dwelling occupancy at minor and major codons, respectively. This larger difference in codons translation velocities alters translation globally and enables kinetically unfavorable processes such as nascent chain deubiquitination to take place. In turn, this leads to abortive translation and favors protein aggregation. These findings highlight the global impact of Not4 and Not5 in controlling the speed of mRNA translation and transition from initiation to elongation. SummaryNot4 and Not5 regulate translation synchronously but distinguishably, facilitating smooth transition from initiation to elongation

molecular biology↗