Summary
Pontocerebellar hypoplasia (PCH), a rare neurodevelopmental disorder, is associated with changes in genes coding for the tRNA splicing endonuclease (TSEN). This multicomponent enzyme is involved in generating essential building blocks for protein synthesis, a process that is crucial to establish and maintain the function of healthy cells. TSEN is found in every cell of the human body but the effect of the PCH-linked mutations only manifests in specific regions of the brain; a phenomenon not understood at all yet.
Recently, scientists have been able to provide 3-dimensional (3D) structures of TSEN bound to its substrates; so-called transfer RNA precursors or pre-tRNAs. This achievement is crucial for understanding interactions within the complex and with its substrates and allows rationalization of the effects caused by PCH mutations in the TSEN genes.
Protein synthesis and tRNA
Proteins are central building blocks of our bodies, involved in all cellular functions, from assembling cellular structures to metabolic processes and cell divisions. Proteins are made from amino acids and generated in two steps. First, genetic information encoded in DNA is transcribed into messenger RNA (mRNA) in a process called transcription. Second, this intermediate, the messenger, is used as a template to produce proteins in a process called translation. During the process of translation, molecules called transfer RNAs (tRNA) bring the appropriate building blocks (amino acids) to the protein-synthesizing machinery of the cell, where the proteins are generated step by step like a string of pearls. Hence, a sufficient supply of tRNAs is of key importance for protein synthesis and disturbances in tRNA supply lead to cellular stress. Once synthesized, proteins acquire characteristic 3D structures that allows them to fulfil their cellular functions and to specifically interact with other proteins.
tRNA biogenesis and splicing
tRNA biogenesis refers to the process by which tRNAs are created and matured within the cell. This process is highly complex and involves multiple steps, including cutting, folding, and chemical modifications of the tRNA molecules. Proper tRNA biogenesis is crucial for the cell because any errors in this process can lead to defective tRNAs. Because matured tRNAs are the carriers of amino acids that are needed to generate new proteins any interference with their biogenesis is likely to disrupt protein synthesis and overall cellular function.
Some tRNAs have segments that must be removed through a process called tRNA splicing. During this process, these segments, called introns, are excised from the precursor tRNA molecules, while the remaining parts, the exons, are joined together to form mature tRNAs. This is carried out by specialized enzyme complexes, including the TSEN complex.
The TSEN complex is composed of 4 proteins – TSEN2, TSEN15, TSEN34, and TSEN54 – and mutations in these subunits result in different subtypes of PCH. Changes in TSEN54 in particular are linked to three PCH subtypes: PCH2, PCH4 and PCH5. However, the exact mechanisms by which these changes in the TSEN54 gene lead to disease, predominantly affecting specific brain regions, are not yet understood.
Advancements in understanding TSEN biology
A recent study used fibroblasts from skin biopsies of PCH2a-affected individuals and showed that the formation of the TSEN complex in those cells is hampered1. In the test tube, however, the specific activity of the mutant complex could still be shown. The exact reason for this discrepancy needs to be addressed in the future.
To better understand the protein-protein interactions within the TSEN complex and its binding to tRNA, it is crucial to comprehend the complex 3D structures of the proteins at work. Structural snapshots of different states during catalysis can then reveal where certain mutations are located and whether they hamper protein-protein interactions or interfere with catalysis.
Four structures of the TSEN complex at different catalytic states were recently published2345. The structures revealed that PCH-associated mutations are located far from that active (catalytic) sites of the TSEN complex. These findings are important because they enhance our understanding about the complex and its biochemistry. Additionally, these structures also allow us to better understand effects found in other animal or cellular models of PCH, which are essential for identifying biochemical mechanisms associated with PCH.
Conclusion
Alterations in enzyme complexes involved in tRNA splicing, an essential process for protein synthesis, can have severe cellular consequences. However, the mechanisms that link these mutations to the symptoms observed in PCH2 patients remain unclear. Insights into the molecular structure of the TSEN complex helped understand the mechanisms of tRNA splicing and how disturbances caused by mutations alter these fine-tuned cellular processes. These findings will be instrumental for developing future treatment strategies.
References
- Sekulovski, S., Devant, P., Panizza, S., Gogakos, T., Pitiriciu, A., Heitmeier, K., … & Trowitzsch, S. (2021). Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia. Nature communications, 12(1), 5610. ↩︎
- Hayne, C. K., Butay, K. J. U., Stewart, Z. D., Krahn, J. M., Perera, L., Williams, J. G., … & Stanley, R. E. (2023). Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex. Nature structural & molecular biology, 30(6), 824-833. ↩︎
- Sekulovski, S., Sušac, L., Stelzl, L. S., Tampé, R., & Trowitzsch, S. (2023). Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN. Nature Structural & Molecular Biology, 30(6), 834-840. ↩︎
- Yuan, L., Han, Y., Zhao, J., Zhang, Y., & Sun, Y. (2023). Recognition and cleavage mechanism of intron-containing pre-tRNA by human TSEN endonuclease complex. Nature Communications, 14(1), 6071. ↩︎
- Zhang, X., Yang, F., Zhan, X., Bian, T., Xing, Z., Lu, Y., & Shi, Y. (2023). Structural basis of pre-tRNA intron removal by human tRNA splicing endonuclease. Molecular Cell, 83(8), 1328-1339. ↩︎