Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Abstract

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNALU gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNAI"U gene. Extracts from HeLa or CV1 cells transcribed both tRNALeU genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNALu.Surprisingly, when the same tRNAI"U genes were introduced into CV1 cells, only pre-tRNAsLu were produced. The pre-tRNAsI"u made in vivo were of the same size and contained the same 5'-leader and 3'-trailer sequences as did pre-tRNAsLu made in vitro. Furthermore, the pre-tRNAsI"u made in vivo were processed to mature tRNAIeU when incubated with HeLa cell extracts. A tRNALeU gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNALu in CV1 cells is blocked at the level of 5'-and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAslu synthesized in CV1 cells are not transported to the appropriate site.

In vivo pre-tRNA processing in Saccharomyces cerevisiae.

“…This finding suggests that the alternate processing pathway is operative for other pre-tRNA families. Our interpretation is supported by previous observations in that HeLa cell extracts were shown to splice some end-extended, intron-containing pre-tRNAs (16,56,62). Since end processing need not precede splicing of introncontaining pre-tRNAs, these events are not related in a dependent order.…”

Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs.

“…UAAUUG and pppAAG, both migrating closely together. Hence, transcription starts at the A residue at position -8 (Ganguly et al, 1988). [a-32P]UTP labelling of the pre-tRNAs followed by fingerprinting (not shown) revealed that transcription termination in both cases occurs in stretches of 7 and 8 consecutive T residues, respectively, which almost immediately follow the end of the 3' exon (Filipowicz and Shatkin, 1983;Ganguly et al, 1988).…”

“…Characterization of in vitro synthesized yeast and vertebrate tRNA precursors For studying in vitro splicing of heterologous pre-tRNAs in a wheat germ extract we selected tRNA genes for which efficient transcription in HeLa cell extracts was known, a prerequisite for our studies since an in vitro polymerase III transcription system from plant cells does not yet exist. Transcription in HeLa cell extracts has been established for the two yeast tRNAGA and tRNALu genes (Filipowicz and Shatkin, 1983;Ganguly et al, 1988), for the Xenopus (Laski et al, 1983;Gouilloud and Clarkson, 1986) and the human tRNATYr gene (van Tol et al, 1987). We had planned to include several yeast tRNATyr genes in our studies since tRNATYr genes were also available from Nicotiana, Xenopus and man.…”

“…Aliquots of the 32P-labelled pre-tRNAs were incubated for 120 min in the wheat germ S100 extract and the resulting RNA species YS104, YL117, X89, and H96 (seen in Figure 2) were also analysed by fingerprinting as described above. The oligonucleotides were identified by their position on the fingerprint (Domdey et al, 1978) and by comparison with the corresponding DNA sequences (Olson et al, 1981;Laski et al, 1983;van Tol et al, 1987;Ganguly et al, 1988). Oligonucleotides specific for the 3'-and 5'-flanking sequences are underlined, those deriving from the intron are dotted.…”

“…Escherichia coli JM 109 was used as a host for propagation of plasmid DNAs. The recombinant plasmids used in this study were: pPM5 and pAA101, which contain the tRNACGA gene on a 2.5 kb BamHI -HindIllfragment and the tRNALu gene on a 2.2 kb XhoI-SatI-fragment, respectively, from S. cerevisiae DNA subcloned into pBR322 (Olson et al, 1981;Ganguly et al, 1988); pSVtTsu-, a recombinant of pBR322, SV40 and a 263-bp HaeII-HhaI-fragment of X.taevis DNA, which encodes a tRNATYr gene (Laski et al, 1983); pHtTl, which consists of a HaeIIIfragment of 334 bp derived from human DNA, harbouring a tRNATYr gene (van Tol et al, 1987) and pNtTl, carrying a tRNATYr gene on a 3 kb EcoRI-fragment from Nicotiana rustica DNA (Stange and Beier, 1986), both subcloned into a pUCl9 vector.…”