tRNA Splicing Endonuclease: Novel and Essential Function Beyond tRNA Splicing and Subunit interactions

tRNA splicing is a multi-step process in all eukaryotes. In yeast Saccharomyces cerevisiae and vertebrates, the enzyme catalyzing intron removal from pre-tRNA is a heterotetrameric complex (SEN complex) consisting of four subunits: Sen2, Sen15, Sen34, and Sen54. Although the SEN complex is conserved between yeast and vertebrates, the subcellular location where pre-tRNA splicing occurs is not. In budding yeast, the SEN complex is located at the cytoplasmic surface of mitochondria, whereas in vertebrates, pre-tRNA splicing is nuclear. Two-hybrid analyses have shown that Sen2 interacts with Sen54 and Sen15 interacts with Sen34; however, the exact mechanism of how the heterotetrameric subunits assemble at their endogenous location is unknown. To understand why tRNA splicing occurs at the outer surface of mitochondria in yeast, I engineered yeast to mimic the vertebrate cell biology by expressing nuclear localized tRNA splicing endonuclease subunits and tRNA ligase. Utilizing Northern and RT-PCR analyses, fluorescence in situ hybridization, and a reporter for tRNA aminoacylation, the data demonstrate that all three steps of pre-tRNA splicing; intron removal, ligation, and 2′-phosphate transfer, as well as nuclear export of tRNA and aminoacylation occur efficiently in yeast cells lacking endogenous mitochondrially located tRNA SEN. Therefore, the canonical function for tRNA splicing endonuclease can occur efficiently in the nucleus when yeast cells express nuclear splicing proteins. However, because pre-tRNA splicing in the nucleus fails to complement growth defects of cells with defective endogenous mitochondrially located splicing endonuclease, my studies also show that the yeast SEN complex surprisingly serves a novel and essential function in the cytoplasm that is unrelated to tRNA splicing. The novel function requires all four SEN complex subunits and the catalytic core of the complex. Exploring the novel function, the data show that a subset of pre-rRNAs accumulate when the SEN complex is restricted to the nucleus, indicating that the SEN complex moonlights in rRNA processing.To study interaction and assembly of tRNA SEN subunits in yeast, two sets of plasmids were generated. One set encodes nuclear tRNA SEN tagged with mCherry and the other encodes endogenously located mitochondrial tRNA SEN tagged with GFP. By utilizing the plasmids in co-transformation studies, this study confirms previously published two-hybrid interactions between splicing subunits and also demonstrates a homodimeric interaction between Sen15 subunits in vivo for the first time in yeast, consistent with a recent crystal structure study that predicts Sen15 homodimers in humans. These studies demonstrate for the first time that 1) tRNA splicing can occur in the nucleus of yeast cells that express nuclear splicing proteins but lack functional endogenous mitochondrial SEN, 2) tRNA SEN has a novel and essential function that is unrelated to tRNA splicing, and 3) the novel function of tRNA SEN may be involved in rRNA processing, either directly or indirectly. Taken together, these findings suggest that selection for the subcellular distribution of the SEN complex in yeast may reside not in its canonical, but rather in a novel and essential, activity. Additionally, the study has led to a new method for studying complex protein interactions in vivo, validated by proof-of-principle experiments in addition to possibly uncovering yet unknown Sen15-Sen15 interactions. Sen15-Sen15 interactions are not expected to function in pre-tRNA splicing and therefore the putative dimer may also have another function unrelated to tRNA splicing.