Ribosome Shunting in Cauliflower Mosaic Virus

Abstract

A wheat germ cell-free system was used to study details of ribosome shunting promoted by the cauliflower mosaic virus 35 S RNA leader. By testing a dicistronic construct with the leader placed between two coding regions, we confirmed that the 35 S RNA leader does not include an internal ribosome entry site of the type observed with picornavirus RNAs. A reporter gene fused to the leader was shown to be expressed by ribosomes that had followed the bypass route (shunted) and, with lower efficiency, by ribosomes that had scanned through the whole region. Stem section 1, the most stable of the three stem sections of the leader, was shown to be an important structural element for shunting. Mutations that abolished formation of this stem section drastically reduced reporter gene expression, whereas complementary mutations that restored stem section 1 also restored shunting. A micro-leader capable of shunting consisting of stem section 1 and flanking sequences could be defined. A small open reading frame preceding stem section 1 enhances shunting.

RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins

“…However, the translational leader is extremely conserved in sequence and structure among CaMV strains, a feature that likely reflects its fundamental roles in CaMV genome expression. Engineering small sequence or structural alterations in the 35S RNA leader also can have strong detrimental impact on translation efficacy, and consequently, on virus fitness (35,36). Codepletions of host mRNAs predicted as ''sensitive'' vsRNA targets (e.g., using multiple T-DNA insertions) could provide an alternative method to address this issue.…”

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader

“…The impact of the cis-acting elements from both unstructured regions of the CaMV 35S RNA leader (Fig+ 1) was monitored by translation of the CAT-reporter gene placed downstream of mutated versions of the leader+ For these experiments, we used a CaMV 35S RNA leader-cassette expression construct, in which additional restriction sites allow easy introduction of mutations and structural replacements within the leader region (Fig+ 2A)+ Generally, results were similar whether the constructs were analyzed in vivo in transiently transfected plant protoplasts or in vitro in wheat germ extract (summarized in Table 1)+ All mutations within the 59-proximal region (Fig+ 2B) have a strong impact on translation (Fig+ 3A)+ Deletion of two pyrimidine repeats results in moderate repression (by a factor of 2 to 3; pMH176), indicating that they might function as weak translational enhancers+ Deletion of uORF A substantially decreases translation (up to 85% inhibition; pMH174), as could be expected from a previous study on mutated virus in planta and translation analysis performed in vitro, albeit on another CaMV strain (Dominguez et al+, 1998)+ An almost complete deletion of the 59-proximal unstructured domain (pMH119) fully abolishes translation+ These results suggest that: (1) translation or recognition of the uORF A is required for the downstream ORF expression, rather than for structural rearrangements leading to internal ribosome entry site (IRES) formation, as could be hypothesized; (2) the extended stemloop structure might be sufficiently stable to block scanning ribosomes+…”

“…Expression of the CAT-reporter gene, uORF F, and uORF F::HA under control of structural mutations introduced in the CaMV 35S RNA leader+ A: Resolution of in vitro-translated products in 16+5% SDS-PAGE using the Tricine discontinuous buffer system+ Constructs as depicted in Figure 2 and Table 1; translation efficiency and migration of CAT, as well as migration of uORF F (wt, pMH161, pMH162, pMH163, pMH172, pMH215) or uORF F::HA (wt9, pMH1619, pMH1629, pMH1639, pMH1729, pMH2159) are indicated+ B: Immunoprecipitation of the uORF::HA from pMH163, pMH1639, and pMH2159 translation in vitro+ (Fütterer et al+, 1996)+ Expression of reporter genes under the influence of the shunt-competent CaMV and RTBV pregenomic RNA leaders has been studied in a broad range of experimental conditions: in transiently transfected plant protoplasts (Fütterer et al+, 1993), in vitro (Schmidt-Puchta et al+, 1997), and in planta (Schärer-Hernández & Hohn, 1998)+ Ribosome shunt occurs in plant tissues and cell cultures derived from both CaMV host and nonhost plants, indicating that shunting does not depend on host-specific factors+ In this work, we demonstrate that ribosome shunt occurs on a synthetic mRNA leader just as well as on the viral leader+ Although it has been documented only for a few mRNAs, nonlinear ribosome migration seems to operate in diverse systems and therefore might be considered as a general alternative mechanism for the initiation of translation in eukaryotes+ Nonlinear ribosome migration remains a rather poorly described mechanism for translation initiation in eukaryotes+ Extensive studies on the CaMV pregenomic 35S RNA have shown that ribosome shunt depends exclusively on cis-acting elements in the leader: a lowenergy, elongated hairpin structure preceded by a short ORF terminating upstream (Dominguez et al+, 1998;Hemmings-Mieszczak et al+, 1998;Pooggin et al+, 1998)+ In some of the other cases where shunting has been reported, stable stems also seem to be involved (Füt-terer et al+, 1996;Li, 1996;Yueh & Schneider, 1996)+ Based on the common structural features of the known viral shunt-competent leaders, a synthetic mRNA leader was designed, where the viral structure is precisely replaced by a short, low-energy hairpin+ This particular stem (Fig+ 5A) has been previously used as a device to efficiently interfere with scanning: too stable to be unwound by the scanning complex, it does provoke the 40S subunit stalling on the 59 side of the hairpin (hp7; Kozak, 1989a)+ Incorporation of the artificial hairpin within the viral flanking sequences indeed fully blocks ribosome scanning, but promotes efficient shunting (Table 1; pMH192, pMH193, and pMH195)+…”

“…The best-studied artificial structured leader, pMH188, retains all the features of the original CaMV 35S RNA leader in our translation assays in vivo and in vitro+ In both cases translation is cap dependent and stimulated by cis-acting elements in the 59-proximal region (compare constructs pMH119, pMH176, and pMH212)+ The termination of uORF A in front of the stem structure seems to be of special importance: its deletion abolishes translation on both the viral and the synthetic leader (pMH174 and pMH191)+ In contrast, the role of the 39-flanking sequence is negligible, as even large deletions in this region are well tolerated (pMH187 and pMH200)+ By analogy to the viral IRES structure and function (Pilipenko et al+, 1992;Scheper et al+, 1994), it was previously hypothesized that the 39-proximal pyrimidine-rich sequences could form a "shunt acceptor site," that is, a landing pad for the shunting ribosomes (Fütterer et al+, 1993;Dominguez et al+, 1998)+ In this light, the latter result is rather surprising and suggests that the first AUG codon downstream of the stem might be a proper recognition signal for the ribosomes that have just accomplished shunting+…”

Continuous and Discontinuous Ribosome Scanning on the Cauliflower Mosaic Virus 35 S RNA Leader Is Controlled by Short Open Reading Frames

“…It does not function as an internal ribosome entry site when placed between two cistrons (22). Expression downstream of the 35 S RNA leader occurs via ribosome shunt, which requires three cis elements within the leader, a capped 5Ј end (14,23), a 5Ј proximal sORF (sORF A; 4 codons in length), and, following it after 6 nt, a stable stem (stem section 1) (22,24,25). The translation event at the sORF is needed for efficient shunting (26,27).…”