EAGER: Measuring transcriptome-wide translation initiation rates from a single experiment

Project: Research project

Project Details

Description

Translation is the process by which ribosomes read and use the genetic information in mRNAs to generate proteins with specific cellular functions. The translation initiation rate is the key measure of translational output, as this rate specifies how much protein can be made from each mRNA and hence how much of a particular protein will be available in a cell. The aim of this project is to examine and optimize a novel approach for determining translation initiation rates at a genome-wide level. This approach will allow transcriptome-wide translation initiation rates to be determined quickly and efficiently in a single experiment and for any type of cell. Ultimately, this technique will allow researchers to address fundamental questions about the process of translation more broadly and deeply. The staff exchanges between the theoretical and experimental laboratory groups will allow graduate students and post-doctoral scholars to learn complementary experimental and theoretical skills that will enhance their training as quantitative biologists and biophysicists.

Ribosome profiling is widely used to obtain high-resolution profiles of in vivo translation. Typically, ribosome profiling experiments are carried out in parallel with RNA sequencing experiments on the same sample. The resulting ribosome profiling and RNA sequencing data, however, are not quantitatively comparable due to substantial technical differences in library generation and the lack of an absolute standard permitting normalization between these two independent experiments. This conventional approach also doubles the amount of sequencing required and introduces additional statistical uncertainty in comparing the generated DNA libraries. In this EAGER proposal, the theoretical and experimental expertise of the investigators will be combined to create a new experimental protocol to quantitatively measure messenger RNA abundance and translation from a single experiment, and thereby quantify absolute transcriptome-wide in vivo translation initiation rates. This new experimental protocol will be a variant of ribosome profiling by combining features of RNA sequencing and ribosome profiling into one experimental method. This eliminates the technical differences encountered in comparing the independent experiments of ribosome profiling and RNA sequencing thus allowing direct quantification of transcriptome-wide translation initiation rates. This technique will go beyond conventional qualitative measures of translation control, such as 'translation efficiency', and measure quantitative rates of protein production in vivo.

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.

StatusFinished
Effective start/end date5/1/194/30/23

Funding

  • National Science Foundation: $299,950.00

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