Biology subjects
Publications and source records attributed to Upton, H. E..
Withdrawal statementThe authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2024/592122. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: https://doi.org/10.1101/2024.05.01.592122.
Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RTs dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11-12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.
Ribosome profiling has unveiled diverse regulations and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected mRNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with Ordered Two-Template Relay, a single-tube protocol for sequencing library preparation that incorporates adapters by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved a myriad of distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling, and termination. Our optimized methods for mRNA footprint generation and capture provides a richer translatome profile using lower input and fewer technical challenges.