Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex

Abstract

The splicing of transfer RNA (tRNA) introns is a critical step of tRNA maturation, for intron-containing tRNAs. In eukaryotes, tRNA splicing is a multi-step process that relies on several RNA processing enzymes to facilitate intron removal and exon ligation. Splicing is initiated by the tRNA splicing endonuclease (TSEN) complex which catalyzes the excision of the intron through its two nuclease subunits. Mutations in all four subunits of the TSEN complex are linked to a family of neurodegenerative and neurodevelopmental diseases known as pontocerebellar hypoplasia (PCH). Recent studies provide molecular insights into the structure, function, and regulation of the eukaryotic TSEN complex and are beginning to illuminate how mutations in the TSEN complex lead to neurodegenerative disease. Using new advancements in the prediction of protein structure, we created a three-dimensional model of the human TSEN complex. We review functions of the TSEN complex beyond tRNA splicing by highlighting recently identified substrates of the eukaryotic TSEN complex and discuss mechanisms for the regulation of tRNA splicing, by enzymes that modify cleaved tRNA exons and introns. Finally, we review recent biochemical and animal models that have worked to address the mechanisms that drive PCH and synthesize these studies with previous studies to try to better understand PCH pathogenesis.

Structural Basis for pre-tRNA Recognition and Processing by the Human tRNA Splicing Endonuclease Complex

“…4). Three of the four TSEN subunits contain large, disordered regions that are not visible in the reconstructions 5 . Further, we did not observe well-ordered density for the CLP1 kinase in the reconstruction, suggesting that it either associates with the complex through a dynamic/disordered region, or it dissociates from the complex during cryo-EM grid preparation.…”

“…Previous work has established the significance of the PBP in mediating cleavage at the 3¢ splice site 28,35 . In Drosophila and human intronic tRNAs, this base pair is strictly a C-G, and the strength of the PBP impacts cleavage 4,5,28 . Analogous to the EndA•BHB structure, we observe that the PBP forms an A-minor interaction with the last nucleotide in the 3¢ bulge, A53 (Fig.…”

“…We mapped known PCH disease-associated mutations onto our TSEN structure, with the exception of TSEN54(A307S) which lies in a disordered region of TSEN54 not in the structure (Figure 4A) 5 . Interestingly, none of the mutations appear within active site regions of TSEN34 or TSEN2, but instead are found on surface exposed interfaces that may be sites of interaction with other proteins or in regions that appear important for complex stability.…”

“…Within the human genome, there are several isodecoder families that all contain introns; thus tRNA splicing is essential 3,4 . Eukaryotic tRNA intron cleavage is catalyzed by the heterotetrameric TSEN complex 5 . The TSEN complex contains two presumptive structural subunits, TSEN54 and TSEN15, along with two endonuclease subunits, TSEN34 and TSEN2, that catalyze cleavage at the 3' and 5'-splice sites, respectively 6,7 .…”

“…In vitro reconstitution of with the human TSEN complex has established that CLP1 is not required for intron removal 12,17 . In vivo, CLP1 likely plays an important regulatory role in tRNA splicing by inhibiting exon ligation and/or targeting exonucleolytic decay of the intron 2,5,17 . Mutations in CLP1 and all four TSEN subunits are associated with multiple subtypes of the PCH family of inherited neurodevelopmental disorders [18][19][20][21] .…”