Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease

Abstract

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5[prime] leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRNAArgs containing a 13-nt 3' trailer and a 5[prime] leader of various lengths. The 3' processing was slightly stimulated by 5[prime] leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths. We also investigated whether 3'tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem. Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.

tRNA biology charges to the front

“…From the cocrystal structure (Li de la Sierra-Gallay et al 2006), it is evident that the tRNA substrate binds one subunit and is cleaved by the other subunit, that the protein binds tRNA through numerous contacts in the tRNA backbone, and that the D-stem and anticodon stem are largely devoid of contacts, which is consistent with biochemical data that these regions are not important for activity (Nashimoto et al 1999a). From modeling and biochemical data, it is likely that the 39 trailer binds through an exit tunnel that precludes basepairing (Nashimoto et al 1999b;de la Sierra-Gallay et al 2005;Redko et al 2007), and that the anti-determinant function of the CCA end of RNase Z is due to the loop between strands b1 and b2 and to the flexible arm, also called the exosite (de la Sierra-Gallay et al 2005;Redko et al 2007;Minagawa et al 2008).…”

A Pathogenesis-associated Mutation in Human Mitochondrial tRNALeu(UUR) Leads to Reduced 3′-End Processing and CCA Addition

“…Since a 5 0 -leader can interfere with the cleavage reaction at the 3 0 -end, 15,29,30 the transcripts were synthesized with a mature 5 0 terminus. Cleavage efficiencies of internally labeled transcripts were tested using a partially purified human mitochondrial 3 0 -tRNase.…”

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

“…Although inhibition of 3 ′ cleavage when RNase P processing is reduced has been observed repeatedly (Lee et al 1991;Lygerou et al 1994;Kufel and Tollervey 2003), the reverse has not been reported. Moreover, tRNase Z has been shown to efficiently cleave only 5 ′ -processed pre-tRNAs, and human tRNase Z activity in vitro is inhibited by the presence of longer 5 ′ extensions (Kunzmann et al 1998;Nashimoto et al 1999;Schierling et al 2002;Dubrovsky et al 2004). In turn, our data suggest that the roles of Rex1 and Rex2 proteins and Rrp6 in generating mature tRNA 3 ′ ends do not fully overlap, but that Rrp6 together with Rex2 may also affect the kinetics or efficiency of the 5 ′ leader cleavage by RNase P. It is difficult to explain the basis of this effect, but the existence of a link between 3 ′ and 5 ′ tRNA processing is quite realistic, being manifested when some aspects of end maturation and/or tRNA quality control are defective.…”

“…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).…”