| Summary: | Protein translation has emerged as an important mediator of cellular activity. Here, we discuss efforts to develop and apply new technologies designed to gain insights into translational control. We begin with the application of ribosome profiling to a RiboTag Glioma mouse model which enables translational profiling of transformed cellular populations. This approach demonstrates a number of abnormalities of translation in transformed cells. We go on to report the development of an inexpensive and rapid library preparation methodology which enables high-throughput sequencing of ribosome-protected footprints from small amounts of input material. We apply this technique to a CAMKII RiboTag mouse model to make new insights into cell-type specific translation. Finally, we describe efforts to investigate translation regulatory networks through the development of a technique which couples large-scale perturbation with a genome-wide readout of translation.
Molecular dissection of tissues through the ectopic expression of modified ribosomal proteins commonly relies on tissue-specific genes which act as drivers. In the case of glioma, a gene specific to transformed tissue, but not expressed in normal brain tissue, has not been identified. Chapter 2 focuses on efforts to bypass this through the development of a RiboTag Glioma mouse model which allows for concurrent transformation and the expression of an epitope-tagged ribosomal protein in virally infected cells. This model made possible the isolation of translating mRNA from transformed cellular populations and was used to demonstrate the existence of a number of translational abnormalities in transformed cells.
Conventional ribosome profiling is a powerful tool which allows for the identification of ribosome-protected mRNA footprints. However, it is time-consuming, expensive, and difficult to implement. Based on our experiences with conventional ribosome profiling, we sought to develop a method which could decrease the overall number of enzymatic reactions and purification steps, thereby reducing the time and cost associated with the procedure; these efforts are discussed in Chapter 3. Utilizing a ligation-free library preparation process, which incorporates poly(A)-polymerase, template switching and bead-based purification, we reduced the time, costs and input requirements required to generate a ribosome profiling library while maintaining high library complexity. We applied our ligation-free ribosome profiling technique to a CAMKII RiboTag mouse model which enabled us to identify patterns of cell-type specific translation and the effects of mTOR inhibition in CAMKII-expressing excitatory neurons.
Regulation of protein expression is an essential and highly complex cellular activity. Aberrations of translational control are central to a host of pathologies and have direct clinical relevance. However, our knowledge of the networks which control translation is limited. Chapter 4 details our efforts to develop a highly-scalable technology which enables the identification of gene-specific translational alteration in response to perturbation. Coupled with a large-scale perturbation screen, this technique could lead to the generation of a network for translational control, similar to efforts previously undertaken to understand transcriptional control. By combining the recently developed PLATE-Seq method, which utilizes unique barcode identifiers and pooled library construction, with a technique for the identification and isolation of ribosome associated mRNA, we are able to rapidly and inexpensively determine genome-wide translational states in a highly scalable
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