Kalekar, R. L. | bioRxiv Science

bioRxiv2026

The Translatome of Senescent Cells Revealed by Ribosome Profiling

Cellular senescence drives aging-related tissue dysfunction through the senescence-associated secretory phenotype (SASP), an inflammatory secretome linked to retrotransposable element (RTE) derepression. Transcriptomic and proteomic approaches have extensively characterized the senescence program, but key gaps remain: transcript abundance is a poor proxy for protein output, limited proteomic depth misses low-abundance proteins, and the highly repetitive sequences of RTEs compromise locus-level peptide attribution. To bridge these gaps, we used AHARIBO (AzidoHomoAlanine-mediated RIBOsome isolation), which captures actively translated full-length mRNAs, to profile the translatome of proliferating, senescent, and late-senescent human fibroblasts. Comparing these ribosome-associated transcripts with the total mRNA pool revealed marked post-transcriptional regulation of key senescence programs. Inflammatory SASP components were translationally depleted in senescence, and these transcripts were enriched for AU-rich element-binding protein motifs, including the ZFP36 family, implicating these proteins in post-transcriptional gating of inflammatory signaling. Transcriptome-wide, translational efficiency was associated with 3UTR GC content and specific RNA-binding protein and microRNA (miRNA) motifs. We also observed a striking wobble-position codon bias: a proliferation-specific program favoring A/U-ending codons collapsed in senescence, disproportionately affecting cell-cycle and proliferation gene sets. By pairing AHARIBO with a sample-specific reference genome incorporating non-reference L1 insertions, we resolved translation of individual L1 loci and identified two intact L1HS elements with sustained activation in senescence. One of these, L1HS_14q23.2_3, independently identified in multiple experiments, emerges as a candidate intact L1 locus for producing inflammatory cDNA species. These findings implicate translational control as an important regulatory layer shaping the senescent program.

cell biology↗

bioRxiv2020

Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer

Bulk transcriptomic studies have defined classical and basal-like gene expression subtypes in pancreatic ductal adenocarcinoma (PDAC) that correlate with survival and response to chemotherapy; however, the underlying mechanisms that govern these subtypes and their heterogeneity remain elusive. Here, we performed single-cell RNA-sequencing of 23 metastatic PDAC needle biopsies and matched organoid models to understand how tumor cell-intrinsic features and extrinsic factors in the tumor microenvironment (TME) shape PDAC cancer cell phenotypes. We identify a novel cancer cell state that co-expresses basal-like and classical signatures, demonstrates upregulation of developmental and KRAS-driven gene expression programs, and represents a transitional intermediate between the basal-like and classical poles. Further, we observe structure to the metastatic TME supporting a model whereby reciprocal intercellular signaling shapes the local microenvironment and influences cancer cell transcriptional subtypes. In organoid culture, we find that transcriptional phenotypes are plastic and strongly skew toward the classical expression state, irrespective of genotype. Moreover, we show that patient-relevant transcriptional heterogeneity can be rescued by supplementing organoid media with factors found in the TME in a subtype-specific manner. Collectively, our study demonstrates that distinct microenvironmental signals are critical regulators of clinically relevant PDAC transcriptional states and their plasticity, identifies the necessity for considering the TME in cancer modeling efforts, and provides a generalizable approach for delineating the cell-intrinsic versus -extrinsic factors that govern tumor cell phenotypes.

cancer biology↗