Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

To gain functional insights into human TSEN/CLP1, we designed an expression vector series based on the MultiBac system that allows combinatorial protein complex production in insect and mammalian cells by utilizing a CMV/p10 dual promoter. Using this system, we were able to assemble and purify functional heterotetrameric TSEN and a heteropentameric complex including the RNA kinase CLP1 from infected insect cells. Individual proteins of the complexes were identified using in-gel digestion and LC-MS/MS analysis. To gain atomistic insights into the molecular architecture of the human TSEN complex, we set out to characterize the TSEN15–34 heterodimer by X-ray crystallography. Despite extensive crystallization trials, full-length TSEN15–34 did not yield any crystals. To define a crystallizable core complex, we subjected the full-length complex to limited proteolysis with subsequent size exclusion chromatography. Primarily, we observed two comigrating polypeptide species. The two polypeptide species were identified using in-gel digestion followed by LC-MS/MS analysis. This led to the identification of the polypeptide species corresponding to residues 23 to 170 of TSEN15 and residues 208 to 310 of TSEN34 covering the predicted conserved nuclease domains.