Differential up‐regulation by tRNAs of ribosome‐inactivating proteins

Abstract

Some plant ribosome-inactivating proteins (RIPs) with RNA-N-glycosidase activity on 28S RNA require, for the inactivation of ribosomes, the presence of macromolecular cofactors present in post-ribosomal supernatants. In the case of gelonin one of the cofactors is tRNATrp lacking one or two nucleotides at the 3'-CCA end [Brigotti, M., Carnicelli, D., Alvergna, P., Pallanca, A., Lorenzetti, R., Denaro, M., Sperti, S. and Montanaro, L. (1995) Biochem. J. 310, 249-253]. In the present study it is shown that tRNAs are involved in the up-regulation of all the cofactor-requiring RIPs up to now identified (agrostin, barley RIP, PAP and tritin, besides gelonin). Polyacrylamide gel electrophoresis shows that tRNA fractions with different mobilities stimulate different RIPs. With the identification of agrostin, the cofactor-requiring RIPs (italics) add to five out of a total of thirteen investigated: barley RIP, bryodin-R, gelonin, lychnin, momordin, momorcochin-S, PAP, saporin-6, tritin [Carnicelli, D., Brigotti, M., Montanaro, L. and Sperti, S. (1992) Biochem. Biophys. Res. Commun. 182, 579-582], agrostin, luffin, trichokirin and trichosanthin (present study).

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

“…An assay of the gelonin-stimulating activity of tRNAs was performed in the two-step assay designed for testing the cofactor requirement of RIPs (Carnicelli et al+, 1992;Brigotti et al+, 1995b)+ Briefly, 80S Artemia salina ribosomes (10 pmol in 10 mL) were preincubated with 5 nM gelonin (Sigma), a concentration of the RIP that is inactive in the absence of cofactors, in the presence of 1 mM ATP, and different concentrations of tRNA (up to 2+5 mM)+ After 10 min at 28 8C, 2+5-mL samples were withdrawn and the extent of inactivation of ribosomes was quantified in a 100 mL poly(U) translation system+ The effective dose 50 (ED 50 ), that is, the concentration of tRNA that stimulated 5 nM gelonin to give 50% inactivation of ribosomes, was calculated from the linear regression between ribosome inactivation and log of the concentration of tRNA+ A weight of 24 kDa was assumed for all the tRNAs assayed+…”

“…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+…”

tRNATrp as cofactor of gelonin, a ribosome‐inactivating protein with RNA‐N‐glycosidase activity features required for the cofactor activity

“…In Panel A the binding of trace mounts of [32p]tRNATrp to gelonin and its binding to ribosomes, both in the absence and in the presence of the RIP, were investigated. In this experiment gelonin was present at a concentration well above that which depurinates ribosomes in the absence of cofactors (17). In Panel B, the binding of trace amounts of []25I]gelonin to unlabelled tRNATrp and its binding to ribosomes, in the absence and in the presence of the cofactor tRNA, were investigated.…”

“…The best known example in human pathology is the infection by human immunodeficiency virus-1 (HIV-1)whose reverse transcriptase has tRNA Lys,3 as primer (14). Since different RIPs are up-regulated by different tRNAs (17), the identification of the cofactor tKNAs can help in a rational choice of the RIP to be used in the immunotoxin targeted to undesired cells infected or transformed by retrovirus.…”

“…The two tgNAs share the discriminator base and most of the anticodon loop, which are considered the identity elements for tryptophanyl-tRNA synthetase (5,20), but their St~luences differ by 24 bases out of 75 (20). These differences should provide the basis for the Artemia salina ribosomes (10 pmol) were preincubated with 5 nM gelonin, a concentration of the RIP well below that required for the inactivation of ribosomes in the absence of cofactors (1,17), for 10 rain at 28°C in 10 Ixl of 10 mM Tris-HCl, pH 7.4, containing 100 mM ammonium acetate, 2 mM magnesium acetate and different amounts (5-300 ng) of each tKNA. ATP, when present, was 1 mM.…”