Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

Area of Science:

• Molecular Biology
• Structural Biology
• Neuroscience

Background:

• The heterotetrameric human tRNA splicing endonuclease (TSEN) complex is crucial for removing introns from precursor tRNAs (pre-tRNAs).
• Mutations in TSEN and the associated RNA kinase CLP1 are implicated in the neurodegenerative disorder pontocerebellar hypoplasia (PCH).
• The molecular mechanisms underlying TSEN-CLP1 assembly, substrate binding, and the structural impact of PCH-associated mutations remain largely unknown.

Purpose of the Study:

• To elucidate the three-dimensional structure of the human TSEN complex bound to pre-tRNAs.
• To understand the molecular basis of pre-tRNA recognition and cleavage by TSEN.
• To investigate the structural consequences of disease-linked mutations in TSEN.

Main Methods:

• Single-particle cryogenic electron microscopy (cryo-EM) was employed to reconstruct the structure of the human TSEN complex.
• TSEN was analyzed in complex with intron-containing precursor tRNAs (pre-tRNAs).
• Structural analysis focused on protein-RNA interactions and the localization of disease mutations.

Main Results:

• Cryo-EM reconstructions revealed how TSEN recognizes the body of pre-tRNAs through extensive protein-RNA interactions.
• The TSEN complex pre-positions the 3' splice site for catalytic cleavage.
• Disease-associated mutations were found to be located distally from the active site, leading to destabilization of the TSEN complex.

Conclusions:

• This study provides detailed molecular insights into the mechanism of pre-tRNA recognition and cleavage by human TSEN.
• The findings rationalize how mutations linked to pontocerebellar hypoplasia destabilize the TSEN complex, impacting its function.
• The structural data offers a foundation for understanding TSEN-CLP1 function in tRNA processing and its link to neurodegeneration.