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Cracking the Translation Regulatory Code

Periodic Reporting for period 3 - TranslationRegCode (Cracking the Translation Regulatory Code)

Reporting period: 2019-03-01 to 2020-08-31

Regulation of gene expression is central for understanding how the human body works. Regulation of gene expression underlies major changes that occur in our cells in response to numerous physiological and environmental signals. Furthermore, is often deregulated in numerous diseases. Thus, a deep understanding of the molecular mechanisms underlying regulation of gene expression is essential for understanding disease, and future therapy.

Regulation of transcription, the first step of gene expression, i.e. the synthesis of RNA from DNA, has been widely studies for decades. More recently, the scientific community is becoming increasingly aware of the great complexity of regulation at the level of RNA, and more specifically, RNA translation, the process of protein synthesis from RNA. With emerging new RNA therapies and RNA-based drugs, such as the recent drug treatment for the fatal SMA disease, that manipulates post-transcriptional RNA processing, it is becoming more and more important to get a high resolution understanding of post-transcriptional regulatory processes.

In this project we aim to get a genome-wide, high resolution mapping and understanding of regulation at the level of mRNA translation. While many of the current knowledge is about global levels of the regulation, in this project we plan to characterize and understand the regulation for each and every gene in the human genome, in a wide variety of conditions.
During the past period we have been working to establish several methodologies in the lab, which allow us to obtain high resolution, genome-wide measurements of mRNA levels (RNA-seq) and translation levels (Ribosome footprint profiling) at a high resolution. We have established these protocols and subsequence computational analyses pipelines, which allow us to perform big data analysis, to first quantify mRNA levels and translation levels for each gene in the genome, and further extract novel biological information about regulatory processes that occur in the cell.

We have been studying mouse and human cells under various perturbations, in order to understand what govern changes in translation regulation upon external stimuli to cells. Among the avenues we study are: (1) Stress responses, such as oxidative stress, heat stress, ER stress, and others, which cells undergo all the time as part of normal physiology, (2) protein aggregation, stresses that occur and underlie many types of neurodegenerative diseases, and (3) cellular aging, and how stress responses are altered in the aged cells.

In the past period, we have revealed several new translational regulatory processes (see below) that play a major role in the cellular response to external signals.
One such process is a novel way of coupling protein synthesis demands with altered cellular metabolism. We have discovered this novel regulatory mode when studying cellular responses to ER stress, a stress that challenges protein synthesis and folding inside the ER (Endoplasmic Reticulum), a cellular compartment that is a hub for synthesis and processing of a large part of the cell’s proteome. We are currently expanding and generalizing this phenomenon, to characterize the mechanisms that control it, and understand its roles in other stresses and diseases.
Another ER stress related novel phenomenon that we have found is a widespread repression of ER targets during ER stress. We observed this very strongly in many cell types and conditions. This novel translational regulation is specifically enhanced for ER targets, and plays a major role in the cellular adaptation to the stress.

As part of our efforts to understand translation, we have also stumbled upon a novel post-transcriptional regulatory phenomenon that happens to RNA prior to protein synthesis: transcriptional readthrough. Here, we found that upon a variety of stress conditions, cells fail to stop the process of gene transcription, generating aberrantly long RNA species. These readthrough RNAs remain in the nucleus, thereby reducing the overall pool of mature mRNAs. This phenomenon is very widespread, however regulated specifically for distinct gene subsets, which have special signatures (Vilborg et al. Wiesel et al.).

We are currently continuing to perform comparative analyses to further extract novel insights into post-transcriptional and translational regulation that occur for each and every mRNA, and further developing novel methodologies to test how sequences embedded within individual mRNAs govern the regulation of their own protein synthesis.
As stated above, we have already discovered and characterized several novel regulatory mechanisms, some have been published (Vilborg et al. Wiesel et al.) and others are in revision (Gonen et al.) or in preparation. In addition, we have developed two novel methodologies, one computational, to discover and quantify readthrough transcripts from big data (Wiesel et al.) and one experimental, to identify factors that interact with the translational machinery and regulate translation (Meller et al. in revision).
We expect that by the end of the project, we will have a full mapping of the pan-stress response translatome, and deep understanding of sequence-function relationship, within the framework of global and specific regulatory processes that govern mRNA translation in the human cell.