Molecular Cell | The Shi Yigong team of Westlake University has made new progress! Analyze the splicing mechanism of human pre t

Removing Intron from pre tRNA is essential in all three kingdoms of life. In humans, this process is mediated by tRNA splicing Endonuclease (TSEN), which includes four subunits: TSEN2, TSEN15, TSEN34, and TSEN54.

On April 6, 2023, Shi Yigong's team of Westlake University published a research paper entitled "Structural basis of pre tRNA intron removal by human tRNA splicing endonuclease" online in Molecular Cell (IF=19), which revealed the structural basis of human tRNA splicing Endonuclease to remove the introns in the tRNA precursor. This study reported the cryoelectron microscopy structure of human TSEN binding to full-length pre tRNA at an average resolution of 2.94 and 2.88 Å before and after catalysis, respectively.

The human TSEN has an extended surface groove that accommodates l-type pre tRNA. The mature domain of pretRNA is recognized by the conserved structural elements of TSEN34, TSEN54, and TSEN2. This recognition locates the anti codon stem of pre tRNA and places the 30 splice site and 50 splice site at the catalytic centers of TSEN34 and TSEN2, respectively. Most Intron sequences have no direct interaction with TSEN, which explains why pre tRNAs of different Intron can be accommodated and cleaved. This structure reveals the molecular scale mechanism of TSEN pre tRNA cleavage.

Transport RNA (tRNA) is crucial for the flow of genetic information, which allows Ribosome to translate mRNA into proteins. Mature tRNAs are generated from tRNAs precursors (pre tRNAs) through a series of post-transcriptional processing and modification steps. In the three kingdoms of life, for a subset of pre tRNAs, Intron sequences exist and must be removed by splicing. Among the predicted tRNA genes in the Human genome, at least 28 contain Intron with different lengths and sequences. In archaea and Eukaryote, Intron are removed by tRNA splicing Endonuclease (TSENs), and then two released Exon are connected through a multi-step process involving specific tRNA Ligase.

Eukaryote TSEN includes two catalytic subunits TSEN34 and TSEN2, two structural subunits TSEN54 and TSNE15, TSEN34 and TSEN2 cut pre tRNA at 30 splice site (30SS) and 50 splice site (50SS) respectively. In mammals, the polynucleotide kinase CLP1 is co purified with TSEN. Although CLP1 is optional before in vitro tRNA cleavage, mutations in CLP1 and all TSEN subunits have been associated with tRNA metabolic changes and neuropathy.

Mechanism pattern diagram (from Molecular Cell)

Since the discovery of tRNA Intron in the 1970s, extensive biochemical and Crystallography studies have gained considerable understanding of the pre tRNA cleavage of various types of TSENs. In particular, the structure of the TSEN and BHB RNA motif complexes in archaea reveals some key interactions that are necessary for pre tRNA recognition and cleavage. However, the slow emergence of structural information about Eukaryote TSEN severely limits the understanding of the pre tRNA cleavage mechanism. For example, TSEN is believed to use a molecular scale mechanism to recognize the two site cleavage of pre tRNA, but its foundation is still insufficient due to the lack of structural information on the full length pre tRNA bound by TSEN. In addition, it is still unclear how the four TSEN subunits are organized into a complete Endonuclease with two independent Active site.

This study reported two high-resolution structures of human TSEN binding to full-length pre tRNA: one in the pre catalytic state and the other in the post catalytic state. In order to capture the pre catalytic state, the author introduced two missense mutations in TSEN: H255A in TSEN34 and H377A in TSNE. In summary, the structure of the human TSEN/CLP1/pretRNA complex studied in the pre and post catalytic states provides a framework for understanding the mechanism of pre tRNA splicing.