Biology subjects

Zaccara, S.

Publications and source records attributed to Zaccara, S..

5 recordsLinked to original sources

bioRxiv2026

Single residues within the YTHDF intrinsically disordered region control m6A-dependent mRNA decay

Intrinsically disordered regions (IDRs) mediate protein interactions, condensate partitioning, and regulatory control in RNA-binding proteins, yet the residue-level logic underlying IDR function has remained difficult to define using conventional fragment- and truncation-based approaches. Here, we apply cytosine and adenine base-editor screens to map residue-level regulation within the intrinsically disordered region of YTHDF2, a cytoplasmic reader that couples m6A to mRNA decay. By tiling base edits across YTHDF2 in a YTHDF1/YTHDF3-null background, we identify IDR residues that regulate YTHDF2-dependent cellular fitness. Interestingly, we identified residues that limit YTHDF2 decay activity. Their mutation disrupts endogenous protein-protein interactions and generates hyperactive YTHDF2 variants that promote m6A-dependent mRNA decay and alter recruitment to cytoplasmic RNA granules, without affecting intrinsic m6A binding or protein stability. These findings define the residue-level regulatory logic of the YTHDF2 IDR and establish base-editor screening as a strategy to uncover functional mechanisms encoded within IDR regions.

systems biology↗

bioRxiv2025

A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

Relatively few studies have examined the link between SNPs and mRNA translation, despite the established importance of translational regulation in shaping cell phenotypes. We developed a pipeline analyzing the allelic imbalance in total and polysome-bound mRNAs from paired RNA-seq data of HCT116 cells and identified 40 candidate tranSNPs, i.e. SNPs associated with allele-specific translation. Among them, the SNP rs1053639 (T/A) on DNA damage-inducible transcript 4 (DDIT4) 3UTR was identified, with the reference T allele showing a higher polysome association. rs1053639 TT clones generated by genome editing exhibited significantly higher DDIT4 protein levels than AA ones. The difference in DDIT4 proteins was even greater when cells were treated with Thapsigargin or Nutlin, two perturbations that induce DDIT4 transcription. The RNA-binding protein RBMX influenced these allele-dependent differences in DDIT4 protein expression, as shown by RNA-EMSA, RIP, and smiFISH assays. RBMX depletion reduced DDIT4 protein in TT clones to the AA levels. Functionally, TT clones more effectively repressed mTORC1 under ER stress, while AA clones outcompeted TT clones in vitro or when injected in zebrafish embryos. RBMX depletion increased the fitness of TT cells in co-culture experiments. The rs1053639 AA genotype, under a recessive model, correlates with poor prognosis in TCGA cancer data. Key points- Translatome analysis in HCT116 cells revealed allele-specific mRNA translation for 40 SNPs - rs1053639 (T/A) in DDIT4 3UTR showed allelic differences in mRNA localization & protein expression - AA cells showed weaker mTOR inhibition & higher proliferation; AA individuals had poorer prognosis

molecular biology↗

bioRxiv2024

YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response.

Cells have evolved a robust and highly regulated DNA damage response to preserve their genomic integrity. Although increasing evidence highlights the relevance of RNA regulation, our understanding of its impact on a fully efficient DNA damage response remains limited. Here, through a targeted CRISPR-knockout screen, we identified RNA binding proteins and modifiers that participate in mediating the p53 response. Among the top hits, m6A reader YTHDC1 was identified as a master regulator of p53 expression. YTHDC1 binds to the transcription start sites of TP53 and other genes involved in DNA damage response, promoting their transcriptional elongation. YTHDC1 deficiency leads to reduced TP53 expression, and also retention of introns leading to aberrant protein production of key DNA damage factors. While intron retention is dependent on m6A, YTHDC1 favors TP53 transcriptional pause-release independently of m6A. Depletion of YTHDC1 causes genomic instability and aberrant cancer cell proliferation mediated by genes regulated by YTHDC1. Our results uncover YTHDC1 as an orchestrator of the DNA damage response through distinct mechanisms of co-transcriptional mRNA regulation.

cancer biology↗

bioRxiv2019

p53-induced apoptosis is specified by a translation program regulated by PCBP2 and DHX30.

Activation of p53 by the small molecule Nutlin can result in a combination of cell cycle arrest and apoptosis. The relative strength of these events is difficult to predict by classical gene expression analysis, leaving uncertainty as to the therapeutic benefits of Nutlin. Here, we report a new translational control mechanism shaping p53-dependent apoptosis. Using polysome profiling, we establish Nutlin-induced apoptosis to be associated with the enhanced translation of mRNAs carrying multiple copies of a newly identified 3UTR CG-rich motif mediating p53-dependent death (CGPD-motif). We identified PCBP2 and DHX30 as CGPD-motif interactors. We found that in cells undergoing persistent cell cycle arrest in response to Nutlin, CGPD-motif mRNAs are repressed by the PCBP2-dependent binding of DHX30 to the motif. Thus, upon DHX30 depletion in these cells, the translation of CGPD-motif mRNAs is increased, and the response to Nutlin shifts towards apoptosis. Instead, DHX30 inducible overexpression in SJSA1 cells, that undergo Nutlin-induced apoptosis, leads to decreased translation of CGPD-motif mRNAs. Overall, this work establishes the role of PCBP2-DHX30 in controlling the translation of CGPD-motif mRNAs and thus provide a new mechanism to modulate the induction of p53-dependent apoptosis.

molecular biology↗

bioRxiv2018

Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2-O-dimethyladenosine (m6Am), which is found at the first-transcribed nucleotide. A long-standing challenge has been distinguishing these similar modifications in transcriptome-wide mapping studies. Here we identify and biochemically characterize, PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we definitively identified m6Am sites and generated transcriptome-wide maps that are selective for m6Am and m6A. We find that m6A and m6Am misannotations largely arise from mRNA isoforms with alternate transcription-start sites. These isoforms contain m6Am that appear to map to \"internal\" sites, increasing the likelihood of misannotation. Using the new m6Am annotations, we find that depleting m6Am does not affect mRNA translation but reduces the stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Ams function.

molecular biology↗