Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

Abstract

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (AG = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; AG = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes. hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.The scanning model for initiation in eucaryotes postulates three steps in the association of mRNA with 40S ribosomal subunits: binding of a 40S ribosomal subunit-factor complex to the capped 5' end of the mRNA (step 1), followed by linear migration (step 2) which ends when the 40S subunit reaches (usually) the first AUG codon (step 3).Step 1 is facilitated by the m7G cap (29) and a well-characterized cap-binding protein (30).Step 3, recognition of the AUG codon, is mediated by eIF2-modulated base pairing with the anticodon in Met-tRNAMet (5,6) and, at least in higher eucaryotes, by a consensus sequence that flanks the AUG codon (12,14,15). Step 2 is supported by evidence that is provocative but indirect, namely, the effects of the antibiotic edeine on formation of initiation complexes (22) The aforementioned finding emerged in the course of a broader study of the circumstances and cis-limited mechanisms of inhibition of translation by mRNA secondary structure. There have been numerous reports concerning the ability of secondary structure to inhibit translation of eucaryotic mRNAs in vivo (reviewed in reference 17; see also Discussion). Here I have resorted to cell-free translation systems to study the level at which translation is blocked by two specific structures: (i) a moderately stable hairpin (AG = -30 kcal/mol) which does not inhibit translation when it occurs around the AUG initiator codon (i.e., some distance downstream from the cap) but does inhibit when it is moved close to the 5' end of the transcript and (ii) a very stable hairpin (AG = -61 kcallmol) which inhibits, no matter where it occurs within the 5' noncoding region, by halting the migration of 40S ribosom...

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

“…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)+…”

“…In vitro translation products were analyzed in an electrophoretic system (Schägger & von Jagow, 1987) that allows separation of the 4+6-and 7+9-kDa peptides derived from uORF F and the truncated CAT reporter gene, respectively (Fig+ 4A)+ An additional weak band migrating at the position of the expected uORF F product was indeed detected in the case of mutants with decreased stability within stem section I (pMH161, (Fig+ 2A) to replace the natural elongated hairpin structure of the wild-type leader+ All secondary structures used were based on published data demonstrating that an artificial stable hairpin can efficiently block 40S subunit scanning, even if not in direct proximity to the 59-terminal cap structure in the leader (Kozak, 1989a)+ Thus, hp7-type structures flanked by the CaMV 35S RNA leader terminal sequences were tested in translation in vivo and in vitro (Fig+ 3B)+ In both systems, replacement of the CaMV elongated hairpin structure by a short, low-energy stem, in either orientation (pMH188: Ϫ45 kcal/mol; pMH189: Ϫ46 kcal/mol), supports efficient translation, showing that the stem per se, and not its sequence, is recognized by the translation machinery+ Stability of the hair-FIGURE 3. Expression of the CAT-reporter gene under control of mutations in the leader+ A: Mutations in the 59 end (pMH176, pMH174, pMH119), 39 end (pMH150, pMH155, pMH164, pMH165, pMH167 pMH169, pMH177, pMH187)+ B,C: Structural mutations in stem section I (pMH175, pMH160, pMH158, pMH72, pMH163) and an artificial hairpin replacement in the CaMV 35S RNA leader (pMH188, pMH189, pMH191, pMH212, pMH200, pMH190)+ Mutations as depicted in Figures 2 and 5; expression levels in vivo and in vitro are represented by grey and black bars, respectively; resolution of in vitro translated products in 12% SDS-PAGE is shown in the lower panels; c in C indicates control with no external RNA included+ pin in pMH188 seems to be sufficient to support ribosome shunting, as extending the structure does not enhance translation further (Fig+ 3B; pMH190; Ϫ50 kcal/ mol)+ To analyze the mechanism of translation initiation on pMH188, we then introduced additional mutations that were previously found to affect translation under the control of the CaMV 35S RNA leader+ Deletion of uORF A and deletion of the complete 59-proximal unstructured region (Table 1; pMH191 and pMH212, respectively) seriously diminished translation when tested in the context of the pMH188 construct; deletion of most of the 39-proximal unstructured region (pMH200) had only a marginal effect on CAT expression (equivalent to constructs pMH174, pMH119, and pMH187 in the CaMV 35S RNA leader context, respectively; see Figs+ 2 and 5 for sequence of mutants)+ In vitro, as with constructs expressed under the control of the CaMV 35S RNA leader (Hemmings-Mieszczak et al+, 1998), translation on the pMH188 transcript is optimal at 30 8C and a potassium acetate concentration of 100 mM (Fig+ 6A)+ Altogether, the way the artificial low-energy stem supports translation in pMH188 appears similar to the translation mechanism occurring on the CaMV 35S RNA leader+ Generally, expression levels in vivo on the CaMV 35S RNA leader constructs parallel translation data in vitro indicating that our results reflect differences in translation efficiencies (Fig+ 3A)+ Constructs including artificial hairpins were expressed at a similar level in vivo as those under the control of the CaMV 35S RNA leader (Fig+ 3B); in in vitro experiments, however, their expression was slightly higher+ Differences in ionicstrength conditions between the two translation systems might be responsible for this effect+ In any case, when compared between themselves, all constructs with synthetic stems in the leader are expr...…”

“…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...…”

Mineralocorticoid and glucocorticoid receptors in the brain. Implications for ion permeability and transmitter systems

“…with respect to tissue specificity or developmental stages. The 5'UTR sequence is highly conserved in the rat and in the human MR-and GR-cDNA, suggesting a specific function of this region in the regulation of mRNA stability and translational efficiency (Kozak, 1989).…”

An insight into the post‐transcriptional control of gene expression in cell function

“…Once initiated translation is a very efficient process and even fairly stable structural elements in the coding region of the mRNA will not hinder the advancing ribosome until the stop codon is encountered (Kozak, 1989). Stop codon recognition is not simply a default process due to the lack of a corresponding amino-acyl tRNA.…”