Processing of mammalian tRNA transcriptsin vitro: different pre-tRNAs are processed along alternative pathways that contain a common rale-limiting step

Abstract

We have analyzed the pathways and kinetics of processing of mouse tRNA gene transcripts in vitro. Different transcripts are processed along two alternative pathways. The 3' trailer sequence of the tRNA His primary transcript is excised before the 5' leader sequence. In contrast, for the tRNA Gly primary transcript, the 5' leader sequence is excised before the 3' trailer sequence, as has been found for other monomeric eukaryotic tRNA gene transcripts. Computerized analysis of the kinetics of processing indicates that tRNA Asp, tRNA Gly, tRNA Glu and tRNA His transcripts are processed in a substrate concentration-dependent manner and also reveals the existence of a common rate-limiting step, the rate constant of which is equivalent for three of the four transcripts tested. The processing of one pre-tRNA transcript can be competitively inhibited by addition of another pre-tRNA transcript to the processing reaction. The common rate-limiting step is associated with the conversion of the primary transcript to an intermediate and is independent of sequence and the particular processing pathway of the transcript.

3′-processing of yeast tRNATrp precedes 5′-processing

“…In other eukaryotes, reports of 3Ј-processing preceding 5Ј-processing are rare but not unknown. For example, this processing order has been reported for silkworm and wheat germ extracts (Garber and Gage 1979;Arends and Schön 1997) and for some tRNAs in mouse (Rooney and Harding 1986).…”

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

“…We previously found that 5'-mature, 3'-extended chloroplast tRNAPh, was an effective substrate for further 3' maturation (44). Alternate processing pathways have also been observed (30,36), and it was proposed that the order of 5' and 3' processing in vivo may be governed by kinetic rather than by mechanistic considerations (7).…”

“…The tRNA 3' endonucleases from human, X. laevis, and Drosophila cells and from yeast and rat liver mitochondria are specific for 5'-mature forms of those pre-tRNAs which were tested (6,7,14,28,46). In general, however, it is likely that a requirement for 5'-mature substrates is not absolute and that the order of 5' and 3' cleavages may be different for different pre-tRNAs (30,36; discussed in reference 7).…”

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

“…This has been demonstrated, for example, in S. cerevisiae, where mutations in RNase P block maturation of both 5 ′ and 3 ′ ends (Lee et al 1991;O'Connor and Peebles 1991;Furter et al 1992;Lygerou et al 1994). Nevertheless, several exceptions to this order of processing have been reported, including some tRNAs in mouse and wheat germ extracts (Rooney and Harding 1986;Arends and Schön 1997), and in vivo processing of yeast tRNA Trp (Kufel and Tollervey 2003). tRNA splicing and end processing are not strictly ordered, but in yeast wild-type cells, the rate of splicing is slower, and the presence 3 of the 5 ′ leader has been reported to slow down intron removal; therefore, mainly unspliced and end-processed intermediates are detected (O'Connor and Peebles 1991; Kufel and Tollervey 2003;Hiley et al 2005).…”