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

Anda Pitiriciu,Stefan Weitzer,Frank Baas,Pascal Devant,Marie Barth,Samoil Sekulovski,Simon Trowitzsch,Tasos Gogakos,Carla Schmidt,Thomas Tuschl,Katharina Heitmeier,Javier Martinez,Silvia Panizza,Ewan Phillip Ramsay

doi:10.1038/s41467-021-25870-3

Abstract

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15–34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

Highlights

Introns of human transfer RNA precursors are excised by the transfer RNAs (tRNAs) splicing endonuclease tRNA splicing endonuclease (TSEN) in complex with the RNA kinase CLP1
Here we report the recombinant expression, purification, and assembly of functional human TSEN/CLP1 complex, and reveal that TSEN stability and activity are perturbed in pontocerebellar hypoplasia (PCH) patient cells
We show that heterotetrameric TSEN is assembled from heterodimeric TSEN15–34 and TSEN2–54 subcomplexes, which combine to form the composite active sites for catalysis (Fig. 1e)

Summary

Introduction

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. The intron in pre-tRNAs is suggested to allow the formation of a double helix that extends the anticodon stem in the conventional tRNA cloverleaf structure and presents the 5′ and 3′ splice sites in single-stranded regions accessible to TSEN17,18. Such a bulge-helix-bulge (BHB) conformation was postulated to act as a universal recognition motif in archaeal pre-tRNA splicing allowing for intron recognition at various positions in pretRNAs19. Mutations in all four subunits of the TSEN complex have been associated with the development of pontocerebellar hypoplasia (PCH), a heterogeneous group of inherited neurodegenerative disorders with prenatal onset characterized by cerebellar hypoplasia and microcephaly[26,27,28,29,30,31]

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Results

Conclusion