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

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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.