Molecular basis of tRNA splicing by the TSEN complex in health and disease

The DNA of the cell contains all the genetic information required to produce the proteins required to carry out all cellular processes. In order to produce the desired protein, the cell must first interpret this genetic code. This interpretation, or translation, is carried out by the combined efforts of messenger (mRNA) and transfer (tRNA). The mRNA (produced by RNA polymerase II) carries the message to the cytoplasm, where large cellular machinery together with tRNA (produced by RNA polymerase III) translates the code into a protein sequence. Therefore, the function of the tRNA is paramount to expressing the desired gene and maintaining essential cellular processes. However, to reach this functional stage, the tRNA is modified by the action of several processing complexes. Initially, RNA polymerase III produces a precursor, pre-tRNA, which contains flanking sequences that must first be removed before the tRNA can function. Additionally, in 6% of all tRNAs there is an intervening sequence or 'intron' which disrupts the tRNA sequence and must also be excised out. This intron excision is carried out by the TSEN/hClp1 complex in a highly specific and regulated manner. Loss of regulation of this complex leads non-specific cleavage and the generation of toxic tRNA fragments which trigger cell death and manifests in human patients as severe neurodegeneration. Indeed, mutations in the TSEN/hClp1 complex have been associated with the development of prontocerebellar hypoplasia, an early-onset neurodegeneration which causes severe mental retardation and premature death. Despite the importance of this fundamental process to normal brain development the exact mechanism and regulation of this process remains unknown. Therefore we aim to solve the structure of the TSEN/hClp1 complex together with its cognate pre-tRNA substrate to understand how the enzyme specifically recognises and excises the intron. Furthermore, we aim to combine this approach with other biochemical studies with recombinant purified protein samples in order to understand how this action is regulated. Recently, using purified TSEN/hClp1, we have discovered a novel mode of TSEN/hClp1 regulation where a small molecule (ATP, which is pivotal in the cells metabolism) binds to and regulates the specificity of the complex. This suggests a potential regulatory strategy where TSEN uses the levels of this molecule inside the cell to regulate its activity and cell survival under stress conditions. Additionally, understanding this regulation may provide a therapeutic strategy based on modifying TSEN/hClp1 activity for these neurodegenerative conditions.

In eukaryotes, following RNA polymerase III transcription, the precursor tRNA (pre-tRNA) must be processed to produce mature tRNA. One such modification is excision of the intron present in 6% of the total pool of human tRNAs. This is carried out by the 5-subunit endonuclease complex TSEN/hClp1 which specifically cleaves the 5' and 3' splice sites using two independent active sites to liberate the intron. Loss of this regulated cleavage leads to aberrant tRNA fragment production, which sensitises neuronal cells to oxidative stress and increases p53-mediated cell apoptosis. This is relevant to brain development as mutations in subunits of TSEN/hClp1 are associated with early-onset of severe neurodegeneration such as prontocerebellar hypoplasia. Despite the apparent importance of regulated intron excision, the mechanism of action and regulation remains unclear. Recently, we have identified a novel regulatory mechanism where ATP binding regulates the specificity of the 5' splice site active site of TSEN/hClp1 in vitro. We hypothesise that TSEN/hClp1 may detect oxidative stress through changes in intracellular ATP concentrations to regulate apoptosis via tRNA fragment production in neurons, inappropriate activation of which leads to neurodegeneration. Therefore, we aim to understand the mechanism of pre-tRNA recognition and cleavage throughout the structure of unbound and substrate-bound TSEN/hClp1 complex, using cryo-EM and cross-linking/native mass spectrometry. This line of research would give unprecedented detail into the mechanism of tRNA splicing and provide the first study into the molecular basis of TSEN-associated neurodegeneration.

The proposed research fits well with the MRC's strategic plan to improve human health through world-class medical research.
The aims described in this proposal impact on a very fundamental biological process, which is still poorly understood and whose dysregulation leads to severe neurocognitive diseases. We anticipate the putcome of this research to be published in high-impact factor journals.
As the ICR champions golden open access, the results will be available to the entire scientific (and non-scientific) community immediately upon acceptance of the manuscripts. We will also immediately post submitted version of the manuscripts to preprint server for biology (bioRXiv, https://www.biorxiv.org) to make it immediately open to the wide scientific community without further delays.
Beneficiaries will be:

1)Society at large. Supporting and promoting academic research excellence in the central field of molecular and structural bioilogy will help maintaining the UK's world leading role in the biosciences. Outcome of the research could also impact design of novel therapies.

2) Industry: the structural work proposed here will represent a solid scaffold for designing novel TSEN/Clp1 activators and could be exploited by pharmaceutical companies to initiate new studies.

3) Personnel. Training will be provided for a PDRA in modern aspects of structural biology. The PDRA will be trained in modern cryo-EM and microscope operation throughout the LOnCEM consortium. This a very sought-after skill in the job market.

4) General public: we will hosts schoolchildren and the general public to open events, regularly organised by the ICR. We will create a bespoke tour of the facilities and drive the children throughout aspects of modern structural biology, including showing macromolecular complexes in 3D, in order to inspire them becoming the future generation of scientists.