Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases.

Abstract

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed.Synthetic yeast pre-tRNA"te substrates containing the complete CCA7476 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAoH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNAPhe derivative, tRNA-ACAtrailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNAPhe precursors between nucleotides 73 and 74 to give tRNAOH. This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

5′ end maturation and RNA editing have to precede tRNA 3′ processing in plant mitochondria

“…9, 39, 44, and 45 and M. Mayer and A.M., unpublished results), mitochondrion (ref. 4 and this work), and chloroplast (36,40,44), all three enzymes possibly encoded by very similar or even a single nuclear gene(s) and routed to the different organelles after translation in the cytoplasm. It will thus be very interesting to identify the respective nuclear gene(s) in plants and to analyze the sorting of each of the encoded proteins.…”

“…Because in mammalian and yeast mitochondria, 3Ј processing is also performed in a single-step reaction (5,7,43), these organelles most likely lost their prokaryotic 3Ј processing activity and adapted the nuclear host-derived enzyme. A similar process has probably occurred in chloroplast evolution, because these plant organelles also use a eukaryotic-like 3Ј processing pathway (36,40,44). Plant cells appear to contain similar RNase Z activities in each compartment: nucleus (refs.…”

Co-evolution of tRNA 3′ trailer sequences with 3′ processing enzymes in bacteria

“…Endonucleolytic activities that are responsible for eukaryotic tRNA 3Ј processing (3Ј tRNase or RNase Z) have been described in various organisms (Castano et al 1985;Oommen et al 1992;Nashimoto 1997;Kunzmann et al 1998;Levinger et al 1998). Recently, such activities were purified and cloned from plant and archaea, and are shown to be members of the ELAC1/2 family of proteins (Schiffer et al 2002).…”

tRNA 3′ processing in yeast involves tRNase Z, Rex1, and Rrp6

“…However, CCA-less precursors in Bacillus subtilis are processed endonucleolytically by tRNase Z (Pellegrini et al 2003;Wen et al 2005). Conversely, the major pathway for eukaryotic tRNA 3 ′ -end processing is endonucleolytic, while trimming by exonucleases serves as an alternative (Garber and Gage 1979;Hagenbuchle et al 1979;Castaño et al 1985;Engelke et al 1985;Frendewey et al 1985;Manam and Van Tuyle 1987;Stange and Beier 1987;Furter et al 1992;Oommen et al 1992;Papadimitriou and Gross 1996;Han and Kang 1997;Nashimoto 1997;Mayer et al 2000;Schiffer et al 2002).…”