Title: Genome-wide Mapping of Ribosome Occupancies with Inhibitor-Induced mRNA Covalent
Labeling
Ribosome profiling is a powerful method to study translation through genome-wide mapping of
translating ribosomes. This breakthrough technology has illuminated many facets of protein synthesis,
such as ribosomal pausing, alternative initiation sites, and protein synthesis rates across numerous
cellular systems. Although recent innovations have reduced ribosome profiling to single cells, ribosome
profiling typically requires large numbers of cells to conduct and therefore is not routinely used to study
rare cell types and clinical samples. Furthermore, ribosome profiling fragments mRNA and hence
cannot map multiple ribosomes along a single mRNA molecule and requires integration of separate
mRNA sequencing datasets to account for the total number of transcripts. New efficient methods to
simultaneously measure ribosome occupancies and mRNA abundance by preserving mRNA integrity
would further enable the global study of translational regulation and provide new fundamental insight
into translation dynamics. To bridge these technological gaps, here we propose to develop new small
molecule tools based off the natural product ribosome inhibitors to chemically footprint ribosome
positioning atop mRNA transcripts through covalent labeling. Our central hypothesis is that these
mRNA chemical footprints can be detected through error prone reverse transcription and subsequent
mRNA sequencing to map ribosome occupancies while maintaining the integrity of the mRNA. This
approach is termed ribosome mutational profiling (Ribo-MaP). To develop Ribo-MaP, the first aim
proposes the structure-based design of natural product derivatives that potently inhibit ribosome
elongation while maintaining polysome stability. Critically, these derivatives are based off emetine, a
ribosome inhibitor which contacts the mRNA template to block translation. Furthermore, photoaffinity
labeling groups will be attached to these emetine derivatives and the ability to covalently label mRNA
will be tested. Additionally, an alternative approach for mRNA covalent labeling is proposed, in which
emetine derivatives will be functionalized with reactive acylating groups to label the proximal mRNA 2'-
hydroxyl. In both approaches, mRNA labeling efficiency and the ability to readout covalent modifications
as mutations in error prone reverse transcription will be measured. In the second aim, the ability of
Ribo-MaP to map ribosome occupancies genome-wide will be evaluated and benchmarked against
conventional ribosome profiling. We expect that Ribo-MaP will bypass inefficient steps in ribosome
profiling and drop into the streamlined workflow of both robust mRNA sequencing methods and new
emerging technologies for single-cell and long-read mRNA sequencing. Altogether, Ribo-MaP would
provide a highly enabling method to broadly study protein synthesis and ribosome dynamics in cells.