Biology subjects

Roberson, A. B.

Publications and source records attributed to Roberson, A. B..

3 recordsLinked to original sources

bioRxiv2026

Quantitative profiling of basal and stress-induced ribosome collisions

Ribosomes stall when they encounter problematic codons or cellular stress that perturbs translation. Stalled ribosomes can lead to the formation of ribosome collisions, also known as disomes, that engage cellular surveillance and stress signaling pathways. How many disomes form during basal conditions and how disome levels change under stress remain poorly understood. Here, we used spike-in normalized Ribo-seq and Disome-seq to quantify transcriptome-wide disome levels. Applying this approach in yeast and human cells, we found that disomes comprise approximately 2-10% of translating ribosomes under basal conditions. A high-resolution Disome-seq experiment in human cells identified reproducible disome-forming sites that contribute to the basal level of disome formation in the cell. Exposure of yeast cells to methyl methanesulfonate and human cells to anisomycin increased disome abundance up to four-fold and changed the distribution of collisions in a stress-specific and context-dependent manner. Overall, these data provide a quantitative, transcriptome-wide framework for measuring disome levels and reveal how translational stress reshapes the landscape of ribosome collisions in cells.

molecular biology↗

bioRxiv2026

5-Azacytidine incorporation into mRNAs disrupts translation and induces ribosome collisions

5-Azacytidine (5-AzaC) is a cytidine analog and is widely used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although its therapeutic activity is primarily attributed to hypomethylation resulting from DNA incorporation, the majority of 5-AzaC is incorporated into RNA. However, the functional consequences of 5-AzaC incorporation into RNA have been unknown. Here, we show that 5-AzaC treatment of cells leads to inhibition of protein synthesis. Ribo-seq, Disome-seq, and RNA-seq in cells treated with 5-AzaC exhibit a time-dependent C-to-G transversion signature in mRNAs within 2 h of treatment. These transversion events are enriched within footprint positions corresponding to the A-site of monosomes or leading stalled ribosome in a disome complex. Consistently, ribosome and disome footprints are accumulated at sites with C-rich codons in the A-site, specifically with the codons containing a C in the second position. 5-AzaC activates the integrated stress response (ISR) and the ribotoxic stress response (RSR) in a GCN2- and ZAK-dependent manner, consistent with disome-mediated signaling. Furthermore, loss of the Ribosome Quality Control (RQC) factor, ZNF598, sensitizes cells to 5-AzaC. Collectively, our results support a model where 5-AzaC is rapidly incorporated into mRNAs, disrupts decoding, and triggers disome-mediated signaling pathways, which contribute to its cytotoxicity. These findings suggest that translation disruption represents an additional layer of 5-AzaCs mechanism of action, alongside its known DNA-mediated effects.

biochemistry↗

bioRxiv2025

A reversible feedback mechanism regulating mitochondrial heme synthesis

Proper heme biosynthesis is essential for numerous cellular functions across nearly all life forms. In humans, dysregulated heme metabolism is linked to multiple blood diseases, neurodegeneration, cardiovascular disease, and metabolic disorders. Erythroid heme production begins with the rate-limiting enzyme Aminolevulinic Acid Synthase (ALAS2) in the mitochondrion. Although prior studies discuss the regulation of ALAS2 in the nucleus and cytoplasm, its modulation as a mature mitochondrial matrix enzyme remains poorly understood. We report that heme binds mature human ALAS2 with high affinity, acting as a reversible mixed inhibitor that reduces enzymatic activity. Structure-based modeling reveals two flexible regions of ALAS2 interact with heme, locking the enzyme in an inactive conformation and occluding the active site. Our work reveals a negative feedback mechanism for heme synthesis, offering insights into the spatial regulation of ALAS2 and the maturation of the essential heme cofactor.

biochemistry↗