3′-immature tRNATrp is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase

Abstract

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3'-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.

Ribosome‐inactivating proteins from plants: more than RNA N‐glycosidases?

“…The cofactor requirements of the type 1 RIP gelonin (from Gelonium multiflorum) were studied in some detail. One of the supernatant factors for gelonin was identified as tRNA trp (59,60). Only avian (chicken) and mammalian (beef, rat, and rabbit) tRNAs Trp are active, whereas yeast tRNA Trp is completely devoid of stimulating activity.…”

rRNA-like sequences occur in diverse primary transcripts: Implications for the control of gene expression

“…31). The ␣-sarcin sequence is located on the exterior surface of the ribosome and can interact with other RNAs or proteins that affect ribosome function (32,33). The possibility of cellular RNAs that contain rRNA-like sequences interacting with the exterior surface of ribosomes is particularly interesting in light of a recent report in which three antisense sequences were inserted into an expansion segment of the Tetrahymena 28S rRNA that was known to be located on the exterior surface of the ribosome; these modified ribosomes were able to inhibit gene expression apparently at the level of translation (34).…”

Identity elements in bovine tRNATrp required for the specific stimulation of gelonin, a plant ribosome-inactivating protein

“…The 0+5-mL transcription reaction mixtures (Sampson & Uhlenbeck, 1988) contained 2 mg of the amplified DNA, 40 mM Tris-HCl, pH 8+1, 7 mM MgCl 2 , 1 mM spermidine (Sigma), 4 mM dithiothreitol (DTT), 25 mg bovine serum albumin (Pharmacia), 1 mM each NTP (Pharmacia), 4 mM GMP, and 4,000 units of T7 RNA polymerase (Promega)+ Following 2 h incubation at 37 8C and addition of EDTA (50 mM final concentration), the reaction mixtures were extracted once with phenol/ chloroform/isoamylalcohol (25:24:1), once with chloroform/ isoamylalcohol (24:1), and ethanol precipitated+ Precipitates were electrophoresed on denaturating 20% polyacrylamidegels, each giving a single spot+ After elution transcripts were separated from gel impurities by chromatography on Mono-Q columns (HR 5/5, Pharmacia) from which they eluted at 0+8 M NaCl (Brigotti et al+, 1995a)+ Storage of the tRNA transcripts precipitated with ethanol and dissolved in water was at Ϫ80 8C+ All tRNA transcripts retained the ability to accept tryptophan, indicating a proper folding of the molecules, when assayed with aminoacyl-tRNA synthetase from the appropriate source+ Aminoacylation was carried out in 25-mL reaction mixtures (Brigotti et al+, 1998c) containing 100 mM Tris-HCl, pH 7+5, 15 mM magnesium acetate, 0+1 mM EDTA, 10 mM ATP, 1 mCi of [ 3 H]tryptophan (30 Ci/mmol, Amersham), tRNA ranging from 1+5 to 6 pmol, and 40 U of either bovine liver or yeast aminoacyl-tRNA synthetase (Sigma)+ After 30 min at 37 8C, the acid insoluble radioactivity was measured+ Differing from the wild-type yeast tRNA Trp (Brigotti et al+, 1996), the yeast nonchimeric tRNA Trp transcript was not recognized by the bovine enzyme, but was efficiently charged by the aminoacyl-tRNA synthetase from yeast+ Compared to the wildtype tRNAs (1) the bovine nonchimeric transcript was 60% aminoacylated by the cognate aminoacyl-tRNA synthetase; (2) the yeast nonchimeric transcript was 77% aminoacylated by the cognate aminoacyl-tRNA synthetase, a result consistent with that reported by Yesland and Johnson (1993); (3) all transcripts in Figure 2, except transcript B (transcript R in Fig+ 3), were 41-70% aminoacylated by the bovine enzyme; (4) transcripts in Figure 3 were 65-105% aminoacylated by FIGURE 4. Synthetic oligonucleotides used in the construction and in the amplification of native and chimeric tRNA genes containing the T7 promoter+ A,B: the six oligonucleotides used for the construction of nonchimeric bovine and yeast tRNA Trp , respectively, with the phosphorylation sites indicated+ C: the reverse primers used in PCR amplification+ the yeast enzyme+ The greater or lower accepting activity was not related to a higher or lower gelonin-stimulating activity+…”

“…Ribosome-inactivating proteins (RIPs), although also active on DNA substrates (Barbieri et al+, 1994(Barbieri et al+, , 1997Brigotti et al+, 1998a), are best known as RNA-Nglycosidases that inactivate ribosomes by specifically removing a single adenine from a highly conserved loop present in the large RNA of the large ribosomal subunit (for review, see Barbieri et al+, 1993)+ Most RIPs are quite efficient in the absence of added cofactors (cofactor-independent RIPs)+ A small group of them (agrostin, barley RIP, gelonin, PAP-S, and tritin) require instead for maximal inactivation of isolated ribosomes (Carnicelli et al+, 1997) the simultaneous presence of ATP and macromolecular components of the postribosomal supernatant (cofactor-dependent RIPs)+ One of the macromolecular components responsible for the up-regulation of RIPs is tRNA (Brigotti et al+, 1995b(Brigotti et al+, , 1998b)+ The tRNAs involved vary with different RIPs and display a different spectrum of specificity+ In our study with rabbit tRNAs (Brigotti et al+, 1998b) (1) tritin-S (from wheat germ) was almost equally stimulated by different tRNAs; (2) agrostin (from Agrostemma githago seeds) was maximally stimulated by the isoacceptor tRNA Ala with anticodon IGC; (3) barley RIP (from Hordeum vulgare seeds) by the same isoacceptor tRNA Ala and by tRNA Val ; and, finally, (4) PAP-S (from Phytolacca americana seeds) by tRNA Gly + With agrostin, barley RIP, and PAP-S, specificity was not absolute and other tRNAs were not completely devoid of stimulating activity+ In contrast, gelonin, the RIP from Gelonium multiflorum, is uniquely stimulated by tRNA Trp (Brigotti et al+, 1995a(Brigotti et al+, , 1995b(Brigotti et al+, , 1996, a tRNA largely conserved in avian and mammalian cells+…”