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

Abstract

Across all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.

The life and times of a tRNA

“…Further, a recent study that created mouse models with the PCH relevant CLP1 mutations documented changes in tRNA processing intermediates, but these tRNA processing alterations did not correlate with pathogenicity; rather, the pathogenicity correlated with alterations of 3' poly(A) site selection of particular RNAs; thus, the authors suggest that PCH due to CLP1 mutations may result from defects in RNA 3' processing instead of tRNA biology (Monaghan et al 2021). Nevertheless, cryo-EM structures of TSEN in complex with hsClp1 document that TSEN54 interacts with Clp1 (Hayne et al 2022a;.…”

“…Recently, the Trowitzch and the Stanley groups (Hayne et al 2022a;) each obtained high resolution (3.1Å -3.9Å) cryo-EM structures of the human TSEN heterotetramer enzyme in complex with intron-containing pre-tRNAs. The enzyme-substrate complexes were trapped by either modifying the RNA cleavage sites and/or by utilizing enzyme with alterations of catalytic amino acids.…”

“…Importantly, although the reconstituted human TSEN complex can utilize short RNA sequences containing just the intron and the anticodon stem-loop as substrates (albeit with low kinetic activity), the structural analyses show that TSEN54 has extensive interactions with the mature tRNA domain, supporting the earlier model that TSEN54 acts as a molecular ruler to regulate cutting at the appropriate splice sites (Trotta et al 1997). TSEN15 interactions with tRNAs were not resolved, but it is predicted that TSEN15 "mediates interactions with the intron surrounding the 5' splice site" (Hayne et al 2022a).…”

“…The high-resolution TSEN structures provide further information regarding how the TSEN mutations that cause PCH may affect TSEN structure/function. None of the causative alterations lie within catalytically important locations, but rather they disrupt subunit interactions (Hayne et al 2022a;.…”

“…Since the preponderance of studies of eukaryotic SEN indicated that it interacted with the mature tRNA anticodon stem-loop rather than the splice junctions or intron sequences (Reyes and Abelson 1988;Sekulovski et al 2021;Hayne et al 2022a;, and that accurate pre-tRNA cleavage proceeds by a mechanism measuring the length of the anticodon stem (Reyes and Abelson 1988), it was not anticipated that there would be SEN substrates in addition to tRNAs. However, the Xenopus, budding yeast, and reconstituted human tRNA splicing endonuclease can cleave mini substrates in vitro that contain tRNA stem-loop structures (Fabbri et al 1998;Hayne et al 2020), and there is in vivo evidence suggesting that the budding yeast SEN complex has substrates in addition to intron-containing pre-tRNAs.…”