A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation.

Abstract

Both the 68-base 5' leader (f0) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within 0 is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to 0 than to the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between Q and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation. colinear with the genomic mRNA at the 3' terminus; consequently, all TMV mRNAs contain the same 205-base 3' UTR. The TMV 3' UTR is the functional equivalent to a poly(A) tail in that it promotes efficient translation and increases mRNA stability of chimeric mRNA constructs (12). The 3' UTR is composed of two domains. Located at the 3' terminus, a 105-base tRNA-like domain mimics the three-dimensional structure of true tRNAs to such an extent that many tRNA-specific enzymes will also recognize and modify it (reviewed in references 20 and 21). Immediately upstream of the tRNA-like domain is a 72-base domain composed solely of three RNA pseudoknots (40). This upstream pseudoknot domain (UPD) is responsible for the regulation of translation associated with the TMV 3' UTR (12). The fact that the tRNA-like domain, which itself contains two RNA pseudoknots, does not increase translational efficiency (12) demonstrates that the mere presence of RNA pseudoknots within the 3' UTR is not the basis for enhanced translation.To precisely determine the sequences within the UPD that are essential for the regulation of translation, mutations were made throughout the region and their effects on translation of chimeric mRNA constructs were measured in vivo. The 5'-proximal pseudoknot was not required for UPD function. The secondary and tertiary structure and primary sequence at several positions within the 3'-proximal pseudoknot were critical for regulatory function. The higher-order structure of the middle pseudoknot of the UPD may be needed as well. We also demonstrate that protein-binding activity from both wheat germ and carrot extracts specifically recognizes the UPD. The same protein activity also recognized ...

Similarities between the secondary structure of satellite tobacco mosaic virus and tobamovirus RNAs

“…It was shown (Takamatsu et al, 1990) that the pseudoknot of TMV RNA (positions 6260 to 6289) with the stem containing an adenine that formed a bulge is very important for viral multiplication. This pseudoknot, with the adjacent one upstream, were shown to regulate viral mRNA translation and a consensus sequence and structural requirements were proposed (Leathers et al, 1993). The fact that STMV RNA can adopt the same folding in this region lends further support to our predictions and suggests an important functional role for the structure of STMV.…”

“…The stable pseudoknot stalks were also predicted in the long 3'-UTRs of satellite tobacco necrosis viruses (types 1 and 2), and this suggestion was supported by sequence comparisons (Danthinne et al, 1991). It is possible that such long quasi-continuous RNA helices may be important for virus replication (Danthinne et al, 1991;Lahser et al, 1993) and/or mRNA translation (Leathers et al, 1993).…”

Alternative Translation Initiation of Human Fibroblast Growth Factor 2 mRNA Controlled by Its 3′-Untranslated Region Involves a Poly(A) Switch and a Translational Enhancer

“…A few 3Ј translational enhancers have also been characterized in uncapped and/or unpolyadenylated viruses. A pseudoknot-rich domain in tobacco mosaic virus mRNA 3Ј plays a poly(A) tail role, and a translational enhancer that mimics a 5Ј cap has been found in the barley yellow dwarf virus (32)(33)(34). Two additional enhancers have recently been characterized in the 3Ј-UTRs of rotavirus and hepatitis C virus nonpolyadenylated mRNAs (35,36).…”

Replication of tobacco mosaic virus RNA

“…The tRNA-like domain and UPD play roles in increasing RNA stability and in the regulation of translation, as well as acting as recognition sites for the virus RdRp. The TMV RNA 5' cap structure and 3' UPD interact to regulate translation (Leathers et al 1993;Gallie & Kobayashi 1994). A 102-kDa RNA-binding protein that binds speci¢cally to the poly(CAA) region within the 5'-untranslated leader and to the 3' UPD, and is widely conserved in plant species, has been isolated from wheat, and evidence obtained that this binding is important for e¤-cient translation (Tanguay & Gallie 1996).…”