Insights into translational regulation from ribosome profiling data
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Ribosomes carry out protein synthesis from mRNA templates by a highly regulated process called translation. Within the four phases of translation - initiation, elongation, termination and recycling - the focus of translation regulation studies has traditionally fallen on the initiation as the rate-limiting step in protein production. Recent evidence, however, points to the profound importance of regulatory control of elongation during development, neurologic disease, cell stress and even cancer.
Ribosome profiling provides an unprecedented means of studying translational regulation on a global level. It is based on deep sequencing of ribosome-protected mRNA fragments, capturing snapshots of genome-wide translation. However, as with any new experimental technique, biases inherent in the ribosome profiling method are gradually being explored and understood, and serve to inform further refinement of the technique.
In the first part of this thesis I provide a comprehensive overview of the current state of knowledge on translation and its regulation, particularly at the elongation phase. I describe the ribosome profiling technique, data processing and applications to studying translational regulation. Afterwards, I go on to present the results in the form of two scientific papers. First paper tackles the challenge of ribosome profiling data processing, setting the ground work for second paper. The second paper uses improved processing to explore ribosome stalling and its potential regulatory functions.
The first paper presents Shoelaces, a tool for processing and visualization of ribosome profiling data. Here, I demonstrate how streamlining and standardizing processing steps can contribute to better quality and comparability of data for downstream analyses. At the core of this are (1) filtering genuine translating footprints from noise based on periodicity and (2) determining a specific codon being translated by the ribosome thanks to length-dependent offset calculation. Shoelaces automatically selects footprint lengths and offsets, offering a user-friendly graphical interface as well as command line interface for batch processing. By reanalyzing 79 human libraries, I show that Shoelaces retains more quality data than the original manual analyses.
In the second paper, I investigate regulation of translation elongation by ribosome stalling. Utilizing the robust processing technique developed in the first paper, I apply it to process 20 ribosome datasets form yeast, fruit fly, zebrafish, mouse and human. Hypothesising that deep conservation of translation machinery would exist also for biologically significant stall sites, I detect 3293 of these conserved in at least two organisms. I find that proline and negatively charged amino acids are the main contributors to stalling. Furthermore, many of the stall sites are found in RNA processing genes, suggesting that stalling might play a conserved regulatory role in RNA metabolism. The project provides a rich resource for further in-depth studies on conserved stalling and suggests its possible roles in regulation of translation elongation.
Finally, the last part of this thesis consists of conclusive remarks an critical reflection on the impact these projects brought into the field. Here, I point out possible directions for future investigations. Additionally, I include a related paper, on the use of ribosome profiling data of initiating ribosomes in re-annotation of bacterial genomes. Overall, this thesis demonstrates how mining ribosome profiling data can result in biologically meaningful discoveries pertaining to regulation of translation.