Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA

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

“…To date, translation initiation by nonlinear ribosome migration has been reported only for a few viral mRNAs+ Viral multifunctional leaders, usually overloaded with RNA processing or packaging signals, could potentially interfere with the classical ribosome scanning+ Shunting has been described in viruses infecting animal cells, such as Sendai virus (Curran & Kolakofsky, 1988, 1989, adenovirus (Yueh & Schneider, 1996) and budgerigar fledgling disease virus, BFDV (Li, 1996), as well as in plant-infecting pararetroviruses, CaMV (Fütterer et al+, 1989(Fütterer et al+, , 1993, and rice tungro bacilliform virus, RTBV FIGURE 4. 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...…”

“…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+…”

“…In eukaryotic cells, translation initiation usually occurs according to the ribosome scanning mechanism (Kozak, 1989b)+ This classical model assumes that, at first, the 40S preinitiation complex associates with an mRNA at the 59-terminal cap structure and later unidirectionally scans the mRNA leader until the first AUG codon positioned in an appropriate context is encountered, whereupon the 60S subunit joins and protein synthesis starts+ Translation can be strongly affected by cis-acting elements and trans-acting factors that interfere with ribosome scanning in the leader: (1) highly stable stemloop structures mechanically block scanning by stalling 40S subunits on the 59 site of the hairpin (Pelletier & Sonenberg, 1985;Kozak, 1986Kozak, , 1989a; (2) upstream open reading frames (uORFs) reduce initiation downstream due to the apparent low efficiency of reinitiation at subsequentAUG codons (Kozak, 1989b(Kozak, , 1992Hinnenbusch, 1994); (3) RNA-binding proteins associated with cap-proximal binding sites inhibit recognition of the cap structure due to the steric hindrance (Gray & Hentze, 1994;Stripecke et al+, 1994)+ Modified ribosome scanning mechanisms exist, however, that allow relatively efficient translation despite the presence of inhibitory elements in the leader+ Alternative modes of translation initiation have been broadly studied and fall into several categories: leaky scanning (Kozak, 1992), reinitiation (Geballe & Morris, 1994;Hinnenbusch, 1994), internal initiation (Pelletier & Sonenberg, 1988;Kaminski et al+, 1990;Belsham, 1992), and nonlinear ribosome scanning or ribosome shunt (Curran & Kolakofsky, 1988, 1989Fütterer et al+, 1989Fütterer et al+, , 1993Fütterer et al+, , 1996Yueh & Schneider, 1996)+ The cauliflower mosaic virus (CaMV) pregenomic 35S RNA 600-nt leader (Figs+ 1 and 2A) contains all three elements with the potential to strongly inhibit translation: (1) a low-energy elongated hairpin (Ϫ110 kcal/ mol) that has been characterized by enzymatic and chemical probing (Hemmings-Mieszczak et al+, 1997), (2) several uORFs, and (3) a purine-rich cloverleaf structure that interacts with the CaMV coat protein (O+ Guerra-Peraza, M+ de Tapia, T+ Hohn, and M+ HemmingsMieszczak, submitted)+ However, translation initiation downstream of the leader is cap dep...…”

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

“…Translation initiation of a majority of cellular and viral mRNAs begins with the recognition of the m 7 GTP cap at the 59 terminus of the mRNA by cellular factors that associate with the 43S preinitiation complex to recruit the small ribosomal subunit to the mRNA+ Following 59 cap recognition, the 43S preinitiation complex scans the 59 noncoding region (59 NCR) of the mRNA for an AUG codon that is in the appropriate context for the recruitment of the large ribosomal subunit and subsequent initiation of translation (Kozak, 1987(Kozak, , 1989)+ Thus, RNAs possessing long, highly structured 59 NCRs encoding multiple AUG codons in favorable contexts upstream of the initiator AUG or RNAs lacking m 7 GTP cap structures should not be capable of efficient initiation of translation by a cap-dependent, ribosome-scanning mechanism+ RNAs containing such characteristics belong to an expanding group of viral and cellular RNAs capable of initiating translation by alternative mechanisms+ These mRNAs can be divided into two subsets based on the mechanism by which translation is initiated+ The first subset is characterized by the capdependent recruitment of ribosomes followed by nonlinear migration of these ribosomes ("ribosome shunting") to the initiator AUG of the mRNA+ Cauliflower mosaic virus and adenovirus RNAs have been shown to utilize ribosome shunting for the initiation of translation (Futterer et al+, 1993;Yueh & Schneider, 1996)+ The second subset of alternatively translated RNAs is comprised of RNAs translated by a mechanism of cap-independent, direct internal ribosome bind-ing and entry+ This group contains many cellular and viral RNAs including the genomic RNAs of a family of animal viruses, the Picornaviridae (Jang et al+, 1988;Pelletier & Sonenberg, 1988; for reviews, see Jackson & Kaminski, 1995;Stewart & Semler, 1997)+…”