5′‐ and 3′‐sequences of satellite tobacco necrosis virus RNA promoting translation in tobacco

Abstract

SummaryThe RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5Ј cap and a 3Ј poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5Ј-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5Ј region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5Ј 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5Ј 38 nt.

Structure and function of a cap-independent translation element that functions in either the 3′ or the 5′ untranslated region

“…The only mutation (mL3) that knocked out capindependent translation in the 39 UTR but not the 59 UTR (that cannot be explained by compensating, vectorderived secondary structure as in TE ) was the mutation of the loop in SL-III (Loop III)+ The simplest explanation is that Loop III is involved primarily in 39-59 communication, with little or no role in actual recruitment of the translation apparatus+ For this or any other mutation, it might be considered that an RNA stability element, rather than a translation element, has been disrupted+ In the case of mL3, it would be a 39 UTRspecific stability element+ However, we showed previously that deletion of the entire 39 TE had no detectable effect on mRNA stability in vitro (Wang & Miller, 1995) or in vivo (Wang et al+, 1997)+ Therefore, we assume that the mutations introduced here affected functions more directly related to translation+ Thus, the loop of SL-III appears to be involved primarily in 39-59 communication and not directly in ribosome recruitment+ How the TE interacts with the 59 UTR or at least the 59-proximal AUG where initiation takes place, is still unknown+ This awaits analysis of the viral 59 UTR and factors that interact with the 39 TE+ The 59 UTRs of BYDV genomic RNA and subgenomic RNA1 are both compatible with the 39 TE (Wang et al+, 1999), but a vector-derived 59 UTR (Results) or the highly active ⍀ 59 UTR sequence from TMV (Wang et al+, 1997) gave only low levels of cap-independent translation in the presence of the 39 TE+ Thus, there seem to be specific sequences in the BYDV 59 UTRs that participate in communication with the 39 TE, although there is little sequence similarity between 59 UTRs of BYDV genomic RNA and subgenomic RNA1+ The possibility of direct base pairing with the 59 UTR was tested for the 39 cap-independent translation element of Satellite tobacco necrosis virus (STNV) RNA, which is functionally similar, but bears no significant similarity in sequence (Wang et al+, 1997) or predicted secondary structure (Timmer et al+, 1993;Danthinne et al+, 1993) to TE105+ Covariation mutagenesis did not support base pairing between UTRs of STNV RNA (Meulewaeter et al+, 1998)+ Another possibility is that the interaction is mediated by proteins that bind each UTR+ HCV RNA may provide an example of this+ Like BYDV RNA, HCV RNA lacks a cap and a poly(A) tail+ HCV differs by having a complex IRES that spans the 59 UTR and 59 end of the coding region (Reynolds et al+, 1995), but like BYDV, it has an additional sequence (98-nt "X region") in the 39 UTR that enhances cap-independent translation (Ito et al+, 1998)+ Pyrimidine tract-binding protein (PTB) binds both the IRES (Ali & Siddiqui, 1995) and the X region (Ito & Lai, 1997), suggesting that PTB may be involved in 39-59 UTR communication+ However, the X region stimulates translation by only two-to threefold, even in the absence of the internal PTB-binding site+ Thus, it is not mimicking a cap or a poly(A) tail+…”

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

“…pFM191A and pFM191B are described by Meulewaeter et al (3,5). pVE192 is a pGEM-3Z-derived vector containing the cat coding sequence with an NheI restriction site immediately downstream of the stop codon (unpublished results).…”

“…STNV translation requires the translational enhancer domain (TED), which is located within a 120 nt sequence immediately downstream of the coding region. TED stimulates translation of uncapped RNAs synergistically with the STNV leader both in wheat germ and in tobacco (1)(2)(3). Furthermore, it stimulates cap-independent translation autonomously from different positions within the mRNA (5).…”

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans

“…The 39 end of mRNA also participates in translation initiation (Gallie, 1991;Tarun & Sachs, 1995;Jacobson, 1996;Sachs et al+, 1997)+ The poly(A) tail interacts synergistically with the 59 cap in stimulating translation in vivo (Gallie, 1991; Tarun et al+, 1997; Preiss & Hentze, 1998)+ In viral RNAs that lack a 39 poly(A) tail, other sequences in the 39 UTR may stimulate translation (Leathers et al+, 1993)+ The RNAs of barley yellow dwarf virus (BYDV; Allen et al+, 1999) and satellite tobacco necrosis virus (STNV; Lesnaw & Reichmann, 1970) lack both a 59 cap and a poly(A) tail+ The RNAs of these viruses each contain a different sequence in the 39 UTR that confers efficient cap-independent translation on uncapped mRNA (Danthinne et al+, 1993;Timmer et al+, 1993;Wang & Miller, 1995;Wang et al+, 1997;Meulewaeter et al+, 1998)+ BYDV is in the genus Luteovirus of the family Luteoviridae+ Members of the family Luteoviridae have a single stranded, positive-sense RNA genome of 5+6 to 5+7 kb encoding about six open reading frames (ORFs) (Mayo & Ziegler-Graff, 1996;Miller, 1999)+ Viruses in the genus Polerovirus of the family Luteoviridae have a VPg linked to the 59 terminus of the genome (Mayo et al+, 1982;Murphy et al+, 1989), whereas BYDV RNA has neither a VPg (Shams-bakhsh & Symons, 1997) nor a 59 cap (Allen et al+, 1999)+ During its life cycle, BYDV produces three subgenomic RNAs (sgRNAs) that are 39 coterminal with genomic RNA (gRNA) (Fig+ 1) (Kelly et al+, 1994;Mohan et al+, 1995;Miller et al+, 1997)+ The ORFs (1 and 2) in the 59 half of genome are translated from gRNA (Wang & Miller, 1995)+ ORF 2, which encodes the RNA-dependent RNA polymerase, is translated by ribosomal frameshifting from ORF 1 to generate a 99-kDa fusion product (Di et al+, 1993)+ ORFs 3, 4, and 5 code for the coat protein, movement protein, and an aphid transmission function, respectively (reviewed by Miller, 1999)+ All three ORFs are translated only from sgRNA1 (Fig+ 1) (Brown et al+, 1996)+ ORF 4 is translated by leaky scanning (Dinesh-Kumar & Miller, 1993) and ORF 5 by in-frame readthrough of the ORF 3 stop codon (Brown et al+, 1996)+ Subgenomic RNA2 (sgRNA2) may serve as a message for ORF 6 (K...…”