MFB: Probing Collective Ribosome Organization with PolySnap-seq

About This Grant

With this award, the Molecular Foundations for Biotechnology (MFB) Program is funding Dr. Oded Regev from New York University (NYU) and Dr. Jef Boeke from NYU Langone Health to develop a novel experimental method (PolySnap-seq) to probe the positions of ribosomes along individual RNA molecules. Ribosomes are molecular machines that produce proteins by “walking” along RNA and reading the instructions written in it. Despite being critical for life, it is currently not clear how ribosomes organize along an RNA molecule during gene expression. Are they equally spaced along the RNA molecule or do they cluster to form “convoys”? Do they maintain a safe distance from each other or pile up in “traffic jams”? The experimental method uses an enzyme to modify the bases of RNAs that are not protected by the ribosomes, thereby allowing researchers to take a snapshot of the simultaneous positions of all ribosomes along an RNA molecule. As such, the method provides novel insights into ribosome collective behavior and enables new biotechnology applications to, for example, optimize gene expression. This project provides graduate students and postdoctoral fellows with specialized training in method development, data analysis, and machine learning. Finally, an outreach component uses “yeast art” to introduce the general public to biotechnology. The efficiency in which ribosomes translate mRNA into proteins is affected by a complex regulatory logic encoded in RNA sequence and structure. Understanding this logic is required for the rational design of RNA transcripts in biotechnology. Powerful tools like ribosome profiling revolutionized our understanding of how ribosomes are affected by RNA features. Yet, many fundamental questions related to translation remain elusive, such as the minimum separation between ribosomes, the extent of burst translation, and the effect of single nucleotide variants. This research project will develop PolySnap-seq, a novel method for probing in bulk the simultaneous positions of all ribosomes along individual transcripts using an adenosine deaminase enzyme and long read sequencing to identify stretches of mRNA that are not occupied by ribosomes and, therefore, can be efficiently modified to inosine bases. The method will provide detailed transcriptome-wide information on translation, including the separation between ribosomes, how such spacing depends on the distance from the translation initiation site, and the effect alleles and isoforms have on ribosome profiles. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.