Biology subjects

Benhalevy, D.

Publications and source records attributed to Benhalevy, D..

6 recordsLinked to original sources

bioRxiv2026

Gelato streamlines reproducible and auditable assembly of Western blot figures

Western blotting has been widely used for decades, yet figure preparation remains unstandardized, error-prone and difficult to audit. Gelato is a free Fiji/ImageJ plugin that streamlines intuitive figure preparation, logs processing coordinates, provides a straightforward audit platform andenables reproduction from raw images. By making these capabilities simple and accessible, Gelato facilitates robust Western blot reporting, reducing a substantial burden on authors and reviewers while strengthening scientific rigor.

scientific communication and education↗

bioRxiv2025

RBProximity-CLIP Enables Subcellular Mapping of RNA-Binding Protein Interactions at Nucleotide Resolution

RNA-binding proteins (RBPs) enable post-transcriptional gene regulation (PTGR) through specific interactions with RNA molecules, influencing processes ranging from nuclear processing and export to cytoplasmic localization, translation, storage and degradation. A key determinant of PTGR processes is the subcellular compartmentalization of RBPs, which dictates RNA targets they can access and the regulation performed in that environment. To characterize RBP-RNA interactions at subcellular resolution, we developed RBProximity-CLIP. RBProximity-CLIP enables compartment-specific isolation and profiling of individual RBP-RNA interactions by combining APEX2-based proximity labeling and 4-thiouridine-enhanced RNA-protein crosslinking, with sequential RBP- and biotin-affinity purifications. Using this approach, we profiled the RNA targets of three RBPs, AGO2, YBX1, and ELAVL1, across the cytoplasmic, nuclear, and nucleolar compartments, revealing nucleus-specific miRNA-mediated AGO2 targets, as well as subsets of YBX1 and ELAVL1 targets that differ by compartment, yet share identical binding motifs. RBProximity-CLIP enables specific and sensitive detection of compartment-specific RBP-RNA interactomes, thereby providing new insight into spatial gene regulation by RBPs.

cell biology↗

bioRxiv2025

Repurposed hnRNPC binds mature mRNAs and safeguards the mitotic transcriptome

Upon entry to mitosis, RNA metabolism is broadly suppressed. While mechanistic understanding is limited, the mitotic transcriptome is known to be generally preserved to allow the daughter cells an economical and efficient start. We used highly effective cell cycle synchronization to specifically characterize mitotic hnRNPC, an abundant nuclear RNA-binding protein known for intron binding and splicing regulation during interphase. Two density-distinct hnRNPC-RNP populations (low-and high-density; LD and HD) were identified in mitotic cells. RNA-seq analysis combined with fluorescent Cross Linking and Immuno-Precipitation (fCLIP) revealed hnRNPC binding to 17.1% and 8.7% of expressed genes in LD- and HD-complexes, respectively, with most sites mapping to protein-coding genes (77% and 68%, respectively). Mediated by its known cooperative interaction with U-rich motifs, mitotic hnRNPC acquired prevalent interactions with exons, predominantly within the 3 untranslated region of mature mRNAs. Mitotic hnRNPC also retained intron interactions, predominantly in LD-hnRNPC RNPs, which comigrated with both the spliceosome and mono-ribosomes through a density gradient. Interestingly, LD-hnRNPC also interacted with mature mRNAs characterized by short coding sequences, in agreement with mono-ribosome loading. Conversely, HD-hnRNPC, which co-migrated with poly-ribosomes, predominantly interacted with mature mRNA complexes. Downregulation of hnRNPC elicited a global negative effect on the abundance of its mitotic targets. The data points to the global role of mitotic hnRNPC as a stabilizer of pre-mRNA and mRNA. Future studies should provide additional insights related to its multifunctional roles during mitosis.

cell biology↗

bioRxiv2025

The Translational Landscape of Reactive Astrocytes Reveals the Impact of eIF2B-mediated Dysregulation in VWM Disease

A devastating genetic recessive neurodegenerative disorder, Vanishing White Matter Disease (VWMD), stems from mutations in eIF2B--a master regulator of mRNA translation initiation and mediator of cellular stress response. While astrocytes, the brains essential support cells, are known to be central to VWMD pathology, the molecular mechanisms underlying their dysfunction remain poorly understood. Our study reveals that even a mild mutation in eIF2B5 profoundly disrupts astrocyte mRNA translation regulation upon cytokine-mediated activation, affecting nearly one-third of all expressed genes. Through innovative integration of RNA-seq and Ribo-seq analyses using primary cell cultures of astrocytes isolated from eIF2B5R132H/R132H mice, we discovered attempts to compensate for impaired protein production by increasing mRNA levels. However, this compensation proves insufficient to maintain critical cellular functions. Our comprehensive analysis uncovered significant disruptions in cellular energy production and protein synthesis machinery. We also predicted previously unknown defects in cholesterol biosynthesis within mutant astrocytes. Moreover, a meta-analysis of translation initiation scores pinpointed, for the first time, a short list of specific effector gene candidates that may drive disease progression. This powerful combination of transcriptome and translatome illuminates the complex pathophysiology of VWMD and identifies promising new biomarkers and therapeutic target opportunities.

molecular biology↗

bioRxiv2023

Loss of Lamin A leads to the nuclear translocation of AGO2 and compromised RNA interference

In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Furthermore, in SHSY5Y cells, AGO fPAR-CLIP in Lamin A KO cells revealed significantly reduced activity of RNAi. Further exploration of the nuclear AGO interactome by mass spectrometry indicated that AGO2 is in complex with FAM120A, an RNA-binding protein and known interactor of AGO2. By performing FAM120A fPAR-CLIP, we discovered that FAM120A co-binds AGO targets and that this competition reduces the activity of RNAi. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, RNAi impairment, and selective upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.

biochemistry↗

bioRxiv2017

DHX36 binding at G-rich sites in mRNA untranslated regions promotes translation

Translation efficiency can be affected by mRNA stability and secondary structures, including so-called G-quadruplex (G4) structures. The highly conserved and essential DEAH-box helicase DHX36/RHAU is able to resolve G4 structures on DNA and RNA in vitro, however a system-wide analysis of DHX36 targets and function is lacking. We globally mapped DHX36 occupancy in human cell lines and found that it preferentially binds to G-rich sequences in the coding sequences (CDS) and 5' and 3' untranslated regions (UTR) of more than 4,500 mRNAs. Functional analyses, including RNA sequencing, ribosome footprinting, and quantitative mass spectrometry revealed that DHX36 decreased target mRNA stability. However, target mRNA accumulation in DHX36 KO cells did not lead to a significant increase in ribosome footprints or protein output indicating that they were translationally incompetent. We hypothesize that DHX36 resolves G4 and other structures that interfere with efficient translation initiation.

bioinformatics↗