Deciphering Translational Regulation During the Cell Cycle Using Scarce Sample Polysome Profiling and Flow Cytometry

Abstract

mRNA Translation is a highly regulated process and even more so during cell cycle transition wherein the activation or degradation of proteins mediate progression through the various phases. Genome-wide studies including proteomic and ribo-seq approaches show the importance of gene specific translation regulation along with global translation in cell cycle phases. However, the use of synchronisation obscures the accuracy of gene regulation patterns observed. There is also a lack of research data about the influence of mRNA features like UTR length, structure, and composition on it is translatability. Here, we coupled the well-established, high-sensitive polysome profiling method (Scarce sample polysome profiling; SSP-Profiling) with flow cytometry to obtain mildly fixed, unperturbed cells from different phases of cell cycle and evaluated their transcriptome and translatome. We confirm the distribution of light and heavy polysomal fractions with transcriptome but separation of non polysomal fraction along PC1 suggesting distinct variation in non-translated and translated mRNAs. Using differential gene expression, we preliminarily identify novel transcriptionally regulated genes in cell cycle.Furthermore, we characterise groups of translationally regulated gene and cluster them according to their translational trend in cell cycle. Our study establishes a new method for translation study in biologically limited samples with the possibility of coupled flow assisted sorting. It further delineates the translation landscape of cell cycle in unperturbed lymphoblastoid cells.