Systematic analysis of ribosome occupancy and density in the human transcriptome.

摘要

The eukaryotic gene-expression program is regulated at many levels, including mRNA transcription, processing, localization, decay, and translation, each of which must be orchestrated precisely for a cell to function properly. Although genome-wide changes in mRNA abundance have been widely studied under many different conditions in human cells, regulation of mRNA abundance accounts for only part of the regulation of protein abundance, localization, and function. Thus, studying the architecture of translational regulation on a genomic scale will combine with previous data to provide a richer and more complete picture of how the global program of human gene expression is regulated.;In this dissertation, I describe a streamlined method that pools fractions from a sucrose gradient in such a way that comparative hybridizations of the pools to just two microarrays can be used to make global, systematic measurements of two key parameters of translation: (1) Ribosome Occupancy, the fraction of an mRNA species bound by at least one ribosome, and (2) Ribosome Density, the average number of ribosomes bound per 100 nucleotides of coding sequence. I used this method to profile translation of HeLa S3 cells and make the first genome-wide measurements of ribosome density in human cells. I found that independently prepared biological replicates were highly reproducible. I identified similar characteristics of global translation between yeast and human cells, which suggests common features of genome-wide translation in eukaryotic cells.;I also showed that this method can identify significant changes in relative ribosome occupancy and density under different conditions. I observed hundreds of relative changes in ribosome density between mock-treated and rapamycin-treated HeLa S3 cells, which revealed that rapamycin treatment evokes a complex translational response. Furthermore, the method was used in a collaboration to study the respective contributions of translational inhibition and mRNA decay to regulation by the mircroRNA miR-124 in cultured Human Embryonic Kidney (HEK) 293T cells. Both ribosome occupancy and ribosome density were reduced for hundreds of miR-124 targets when HEK 293T cells were transfected with miR-124.;The streamlined method described here can be broadly applied and provides a valuable tool for further investigation of genome-wide translational control.