tRNA retrograde transport in human cells

Proteins form the building blocks of all cells. They are made from amino acids assembled into polypeptide chains by a specialized machinery in the cytoplasm of cells. Transfer RNAs (tRNAs) are short RNAs that transport amino acids to the protein synthesis machinery. Each tRNAs binds a specific aminoacid and recognizes a specific sequence on the messenger RNA, called "codon". Therefore, protein synthesis proceeds in an orderly fashion, whereby the sequence of amino acids is encoded my the messenger mRNA and tRNAs "read" the code.

tRNAs are synthesized in the nucleus, then specialized cellular factors export tRNAs to the cytoplasm, where they participate in protein translation. It was a long-held dogma that tRNAs move in one direction only - from nucleus to cytoplasm. However we and others independently discovered that tRNAs can travel back to the nucleus in an energy-dependent fashion. Nuclear import of tRNAs is conserved from human to yeast cells and it now has the name of "tRNA retrograde transport". Why do tRNAs travel back to the nucleus? There may be several reasons. First, the cell can slow down protein synthesis in response to lack of nutrients to increase its chances of survival. Rapid import of tRNAs to the nucleus can contribute to this metabolic slow down because there is no protein synthesis in the nucleus, hence tRNAs retrograde transport sequesters tRNAs in a different, well separated compartment. Second, some tRNAs become damaged in the cytoplasm and they may hinder protein synthesis. Again, sequestration of damaged tRNAs in the nucleus prevents damage to protein synthesis.

Interestingly, we discovered that some viruses (such as human immunodeficiency virus - HIV) hijack the tRNA retrograde transport pathway to infect cells.

There is clear and emerging evidence of the importance of the tRNA retrograde pathway in regulation of cell metabolism, tRNA quality control and virus infection but little is know about the cellular carrier that transport tRNAs to the nuclei and how the pathway is regulated. Cell metabolism and tRNA synthesis are connected to cell growth, tumour formation and response to environmental stress. Therefore, understanding the tRNA retrograde pathway will advance our understanding of such fundamental cellular processes.

We now plan to identify the carrier that imports tRNAs into the nucleus. We also plan to elucidate the regulation of retrograde tRNA transport and its impact on two cellular pathways important during stress caused by lack of nutrients and invasion by pathogens. We already have evidence that a tRNA carrier is present and have used biochemical approaches to narrow the list of candidates to thirty four. We will use a systematic approach to identify the carrier amongst the thirty four. We shall also test if tRNA retrograde transport is activated by secretion of interferon and during autophagy, two pathways activated by pathogens and other insults.

If the tRNA retrograde transport is implicated in regulation of protein synthesis and activation of critical cellular pathways, then it will be important to be able to agonize or antagonize the tRNA carrier. This will potentially provide an important target to reduce uncontrolled cell growth in cancer, improve cellular barriers to infection and promote cell survival during stress.

We and others have recently discovered that tRNAs are imported back into the nucleus by an active process. tRNA nuclear import is conserved in eukaryotic cells from yeast to human and it is now called the "retrograde tRNA pathway". Its function may be to regulate cellular metabolism, and be a quality control system by importing defective tRNAs in the nucleus for degradation. tRNAs retrograde transport is stimulated by lack of nutrients, heat shock stress and block of protein synthesis by puromycin. We also found that HIV-1 exploits tRNA retrograde transport for infection.
The importance of the retrograde tRNA transport pathway in control of cell metabolism, tRNA biogenesis and viral infection is clearly emerging. However, the carrier that imports tRNAs into the nucleus of human cells is not known. Furthermore, regulation of the tRNA retrograde transport in human cells is poorly understood. This project has two main aims:
1) To identify the carrier mediating tRNA nuclear import in human cells. We have good preliminary data showing that a carrier is required for tRNA retrograde transport. Using biochemical approaches we have selected thirty four candidates and we shall use a systematic approach to identify which one is the carrier.
2) To elucidate the regulation of tRNA retrograde transport in conditions of cell stress resulting in lower protein translation, such as autophagy and the interferon response.
Our research has profound implications to understand control of cell metabolism and tRNA biogenesis. It has the potential to provide a new target to reduce uncontrolled cell growth in cancer, improve cellular barriers to infection and promote cell survival during stress.

Our research will impact:

1) The academic community in several fields. In the field of tRNA and protein synthesis, because this project will advance our understanding of metabolic regulation in human cells via a novel tRNA retrograde transport pathway. In the field of autophagy, because we shall investigate the cross-talk between tRNA retrograde transport and autophagy to modulate cell metabolism. In the field of oncology, because we shall investigate if the tRNA carrier that imports tRNAs in the nucleus is a new target to reduce cell survival in conditions of stress and hypoxia, often found in solid cancers. Conversely, tRNA retrograde transport may help cells cope with stress and prevent aging. In the nuclear import field, because we shall identify a carrier that imports tRNA into nuclei in human cells, which is unprecedented. In the area of interferon response, because tRNA retrograde transport may be a component of the interferon pathway to inhibit protein synthesis and cell metabolism induced by pathogens invasion.

2) The biotechnology industry by providing a novel and well defined potential drug target, the tRNA carrier or a critical co-factor. It might be possible to antagonize the tRNA carrier, making cells less resistant to nutrient deprivation. This may have implications for the treatment of solid cancers where limiting nutrient availability often occurs. Antagonizing the tRNA carrier may also provide a new therapeutic intervention against HIV-1 infection, because tRNA retrograde transport is exploited by HIV-1 to access the nucleus of infected cells. The biotechnology sector is now gaining a greater interest in antivirals that target the interface between host factors and viral components. We have already identified two novel antiretroviral drug targets (capsid and Hsp90), which, combined with our high throughput cell based screening facility, puts us in an internationally competitive position. Indeed, we have recently been awarded an EU FP7 grant within the Innovative Medicines scheme to identify new chemical compounds that target host-pathogen interfaces, in collaboration with a medium-size pharmaceutical company. A further example of successfully targeting the virus/host interface mechanism will increase the awareness of this as an important area for intervention.

3) Training of research scientist. The researcher working on this project will receive a broad training in cell biology of nuclear import, tRNA biology, autophagy, biochemistry and cellular responses to stress. The scientist will be embedded in my lab and exposed to interdisciplinary research on nuclear import modeling and biophysics of the nuclear pore complex. Furthermore, our expertise in molecular virology will provide an interesting perspective in the use of viruses as tools to discover fundamental cellular processes.