Transcriptome-wide RNA accessibility mapping for investigating RNA structure, function, and regulation
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farenhem-farenhem-phd-biology-2026-thesis .pdf
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Author(s)
Farenhem, Kelsey L.
Advisor(s)
Jain, Ankur
Date Issued
February 2026
Publisher
Massachusetts Institute of Technology
Abstract
RNA performs numerous regulatory and catalytic functions essential to cellular life. This functional diversity arises from RNA's ability to fold into complex, dynamic three-dimensional structures. However, understanding these structures and their dynamics within living cells has remained challenging, limiting our ability to uncover how RNA structure drives cellular processes. This thesis addresses this challenge through the development and application of a novel technology that maps human RNA structures at unprecedented resolution and scale.
I first established DMS-TRAM-seq, a high-throughput chemical probing technique that generates robust, transcriptome-wide maps of RNA accessibility. DMS TRAM-seq provided coverage of over 21 megabases of RNA, with secondary structure predictions for over 6000 transcripts. After validating the technique’s accuracy and benchmarking against established methods, I generated secondary structure predictions for the entire human transcriptome, now publicly available at humanRNAmap.wi.mit.edu. Additionally, I identified over 700 highly structured RNA elements throughout the transcriptome, revealing novel regulatory elements involved in diverse cellular processes.
Next, I developed comparative analyses for DMS-TRAM-seq in order to identify RNA structure and accessibility changes occurring upon various cellular perturbations. In oxidative stress conditions, integration with ribosome profiling showed that translation inhibition broadly drives RNAs toward their thermodynamically favored conformations. Additionally, integration with RBP motifs revealed perturbed RBP-RNA interactions and their consequent effects on RNA structure.
Finally, I applied DMS-TRAM-seq to a neuronal model of amyotrophic lateral sclerosis (ALS), where a mutation in the RNA binding protein (RBP) FUS causes abnormal protein localization and RNA processing defects. This analysis revealed structural dysregulation in snoRNAs and scaRNAs resulting from pathogenic FUS mutations, suggesting new directions for investigating snoRNA and Cajal body dysfunction in ALS pathogenesis.
Altogether, this work establishes DMS-TRAM-seq as a powerful tool for interrogating the RNA structurome and its plasticity at transcriptome-wide scale, opening new avenues for elucidating the structural basis of RNA regulation and dysfunction in disease.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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