Tran, L. (BMEB) – Polysome Shadowing: A Long-Read Sequencing Approach to Study Translation

Translation is a central and highly regulated step of gene expression, yet there are few quantitative, high-throughput tools to study translation. Existing methods such as sucrose gradients provide only bulk ribosome counts, while Ribo-Seq offers positional information in the genome but destroys long-range structure and transcript expression information. Because of these limitations, many fundamental questions about mRNA translation into protein remain difficult to assay. In this proposal, I outline my plans to develop a novel technology, deemed Polysome Shadowing, that covalently marks ribosome-unprotected regions of RNA with hyperactive base editors. Because ribosomes protect ~21–30 nt regions of mRNAs, ribosome “shadows” appear as tracts of unedited bases in long-read sequencing. In Aim 1, I will identify ribosome shadows on single molecules by increasing editing efficiency through optimization of dual cytosine and adenosine base editors and statistical modeling. In Aim 2, I will maximize the accuracy of information recovered from highly-edited RNAs by developing a multipass library preparation protocol to generate high-confidence reads. In Aim 3, I will apply the tools I have already developed to examine previously difficult-to-assay paradigms of translational control in the form of viral frameshifting mechanisms. Together, completion of these aims will build an information-rich sequencing technology capable of positioning ribosomes on intact mRNAs while preserving long-range information and establish feasibility to study nascent paradigms.

Host: Liam Tran, Ph.D. Student, Biomolecular Engineering and Bioinformatics 

Advisor: Joshua Arribere