Possibility of Cytoplasmic pre-tRNA Splicing: the Yeast tRNA Splicing Endonuclease Mainly Localizes on the Mitochondria

Abstract

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

“…In vertebrates, tRNA splicing occurs in the nucleus (Melton et al 1980;Lund and Dahlberg 1998;Paushkin et al 2004), whereas it is a cytoplasmic process in yeast, since the SEN complex is located on the outer surface of mitochondria (Yoshihisa et al 2003). Despite the difference in localization, the structure and function of the SEN complex are conserved from yeast to humans (Trotta et al 1997;Paushkin et al 2004).…”

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

“…In contrast, several lines of evidence show that in yeast, pretRNA intron removal occurs in the cytoplasm, not the nucleoplasm. First, the subunits of the heterotetrameric tRNA-specific splicing endonuclease, Sen15, Sen2, Sen34, and Sen54, colocalize with mitochondria (4,5). Tethering of a noncatalytic subunit, Sen54, to mitochondria does not inhibit pretRNA splicing, whereas mutations prohibiting its mitochondrial association cause accumulation of unspliced pretRNAs (4).…”

“…First, the subunits of the heterotetrameric tRNA-specific splicing endonuclease, Sen15, Sen2, Sen34, and Sen54, colocalize with mitochondria (4,5). Tethering of a noncatalytic subunit, Sen54, to mitochondria does not inhibit pretRNA splicing, whereas mutations prohibiting its mitochondrial association cause accumulation of unspliced pretRNAs (4). Moreover, cells with conditional mutations in a gene encoding a catalytic subunit, Sen2, accumulate unspliced pretRNAs in the cytoplasm (4).…”

P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics

“…The budding yeast, Saccharomyces cerevisiae, is well suited to study these processes because primary export can be easily distinguished from re-export in these cells. The characteristic of S. cerevisiae that allows one to distinguish primary export and re-export is the mitochondrial location of the tRNA splicing endonuclease complex (Yoshihisa et al 2003). This complex, which removes the intron from newly transcribed end-processed pre-tRNA, is located on the cytoplasmic surface of mitochondria (Yoshihisa et al 2003(Yoshihisa et al , 2007.…”

“…The characteristic of S. cerevisiae that allows one to distinguish primary export and re-export is the mitochondrial location of the tRNA splicing endonuclease complex (Yoshihisa et al 2003). This complex, which removes the intron from newly transcribed end-processed pre-tRNA, is located on the cytoplasmic surface of mitochondria (Yoshihisa et al 2003(Yoshihisa et al , 2007. Thus, if there is a defect in primary tRNA export, end-processed intron-containing pre-tRNA will accumulate because the nuclear tRNA are unable to access the mitochondrially located splicing complex.…”