Ribosome Shunting in Cauliflower Mosaic Virus
IDENTIFICATION OF AN ESSENTIAL AND SUFFICIENT STRUCTURAL ELEMENT*

Diana Ines DominguezDagger , Lyubov A. Ryabova, Mikhail M. Pooggin, Waltraud Schmidt-Puchta, Johannes Fütterer§, and Thomas Hohn

From the Friedrich-Miescher-Institute, CH-4002 Basel and § ETH CH-8902 Zürich, Switzerland

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cauliflower mosaic virus (CaMV)1 is the type member of the caulimoviruses, a group of plant para-retroviruses (1). In infected plants, two major RNAs are transcribed from the following two viral promoters: the monocistronic 19 S RNA encoding a multifunctional protein that acts as a translational transactivator, and the pre-genomic 35 S RNA (Fig. 1). The 35 S RNA has several functions; it can act as an mRNA for several viral proteins (2) or as a template for reverse transcription (3) and can be alternatively spliced (4); it also appears to be packaged into viral particles (5). In addition, the 35 S RNA is terminally redundant, i.e. polyadenylation signals exist at both ends of the RNA and must be bypassed in the first instance (6, 7). Hence, regulation of CaMV gene expression may take place at the level of splicing (4), polyadenylation (6, 7), transport of unspliced RNA to the cytoplasm (8), and translation (1).


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Fig. 1.   CaMV genome, transcripts, and secondary structure. A, circular map of the CaMV genome S strain. sORFs are indicated by shaded bars and ORFs by shaded arrows in the outer circle. ORF VII (unknown function), I (M, cell-to-cell movement protein), II (ITF, insect transmission factor), III (proposed role in DNA packaging), IV (GAG, structural proteins), V (POL, polyprotein with protease (PRO), reverse transcriptase, and RNase H activity), VI (TAV, translational transactivator and inclusion body protein). The two primary transcripts are shown as arrows along the respective coding region. B, features of the 35 S RNA. Viral ORFs I-VII are shown. A-F, sORFs, PA, polyadenylation signal; SD, splice donor; SA, splice acceptor; ShD, shunt donor; ShA, shunt acceptor; PBS, primer binding site for reverse transcription; (A)n, poly(A) tail. C, secondary structure of the 35 S RNA modified leader (Lm-CAT) as predicted by the "M fold" program (50), minimal energy -162.1 kcal/mol. The sORFs are named and indicated by thick lines superimposed on the structure. The three main stem sections (st1, st2, and st3) are also shown. Numbers represent the start and end positions for each sORF in the Lm-CAT mRNA. The boxed section shows the extent and the structure of the micro-leader.

Two unusual mechanisms are employed by CaMV to translate its RNAs. First, some viral proteins are expressed from polycistronic mRNAs, although mRNAs encoding more than one open reading frame (ORF) are unusual in eukaryotic cells. The expression of a downstream ORF on polycistronic CaMV mRNAs requires a virus-encoded translational transactivator (9, 10). Second, the 35 S RNA contains a leader with certain features that, according to the scanning model for translation initiation (11), would hinder expression of an ORF fused to it. The leader is unusually long (600 nt), contains 7-9 short open reading frames (sORFs) depending on the strain, and folds into a complex stem-loop structure (12-14). Despite these features, translation of an ORF downstream of the CaMV 35 S RNA leader is still possible both in vivo (15-17) and in vitro (18).

A mechanism referred to as ribosome shunting has been proposed to explain how the translational machinery can overcome the barrier imposed by the CaMV 35 S RNA leader. According to this model, ribosomes together with initiation factors enter at the cap site and begin scanning in the 3' direction. However, scanning is not linear, and at some point near the 5'-end ribosomes are transferred to a position further downstream, skipping the central region. This mechanism is not unique to CaMV; ribosome shunting has also been observed with RNAs of rice tungro bacilliform virus (RTBV; see Ref. 19), adenovirus (20), Sendai virus (21, 22), and budgerigar fledgling disease virus (BFDV; see Ref. 23). Nevertheless, very little is known about the molecular mechanism of shunting.

Shunting in CaMV requires the simultaneous presence of regions from both ends of the leader either in cis or in trans (16, 17). The leader forms a large hairpin structure bringing the ends into close spatial proximity (14, 16). We assume that this structure allows the bypass of the central inhibitory region of the leader, with the essential regions at the ends referred to as shunt donor and shunt acceptor. The alternative possibility that these regions would form an internal ribosome entry site (IRES) is unlikely, since reporter gene expression in protoplasts is inhibited by the insertion of an energy-rich stem close to the cap, indicating that entry of the scanning complex at the 5'-end of the mRNA is required (17).

Until now, translation of CaMV and RTBV pre-genomic RNAs has been mainly studied in vivo by transfection of protoplasts with reporter DNA constructs. However, this experimental system has limitations, since the enzymatic activity that is finally measured is the product of transcription, RNA processing, RNA transport and translation, and modifications of the reporter plasmid can affect any or all of these processes. Also, protoplasting of plant cells and transformation of protoplasts might invoke stress reactions that alter translation mechanisms and the preference for certain mRNAs (24, 25). Thus, an additional method, e.g. in vitro translation, is necessary to consolidate and confirm the translational models proposed for CaMV on the basis of the in vivo data. In the in vitro translation system, mRNA is the starting material, and the assay solely measures the translation process.

Recently a wheat germ cell-free system has been described that allowed translation directed by the CaMV 35 S RNA leader (18), and we have used this system to study the shunt mechanism in more detail. By testing a dicistronic construct in which the CaMV 35 S RNA leader is placed between two ORFs, we confirmed that the leader does not promote internal initiation. Translation of a reporter gene fused to the leader was performed mostly by ribosomes that followed the bypass route (shunted) but also, albeit with lower efficiency, by ribosomes that scanned through the whole region. In wheat germ the same shunt acceptor is used as in cruciferae protoplasts. Our results define a shunt donor for the first time. Furthermore, the stem section at the base of the large CaMV 35 S RNA leader stem structure (stem section 1) was defined as the important element in shunting. Together with its flanking regions, this "micro-leader" supports shunting. A short open reading frame preceding stem section 1 acts as a shunt enhancer.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of Clones-- The cloned U1A gene has been described previously (26). U1A-L-CAT was prepared by subcloning the EcoRV-SphI fragment of plasmid LC20 (12), containing the whole leader and the chloramphenicol acetyltransferase (CAT) gene into the SmaI-SphI sites of plasmid pU1A. Plasmids mono-CAT and L-CAT were previously described as pSPC7 and pSPLC20, respectively (18). Briefly, L-CAT contains the SP6 promoter, the whole CaMV leader (strain S), and the CAT gene fused to the AUG of ORF VII. In mono-CAT, most of the leader was deleted except for the last 50 nucleotides starting at the ClaI site (this part does not contain any AUG codon). Lm-CAT is a plasmid with a modified version of the wild-type leader that contains restriction sites to facilitate the introduction of mutations. An XhoI site was created by introducing two point mutations, 50CTCCAT55 to 50CTCGAG55 (numbering according to Ref. 16) and an NheI site by insertion of two bases, 110GCTC113 to 110GCTAGC115. An SpeI site was created at position 165 with two point mutations, 164ACTTCT169 to 166ACTAGT171. 20 nucleotides between the two BglII sites (position 220 to 240 of the wild-type leader) were deleted. The rest of the leader starting at this 3'-most BglII site and the CAT ORF derive from the BglII-Acc65I fragment of pSPLC20. This Lm-CAT modified leader gives about 50-60% of the expression level obtained with L-CAT in in vitro translation. LmF-CAT was made by replacing the ClaI-NcoI fragment of Lm-CAT with an oligonucleotide carrying a deletion of A592 to create an F::CAT fusion protein. Plasmids (LAUG-free, LonlyA, LonlyF, and LonlyA-F)-CAT contain mutations in the sORF AUG codons (see Fig. 8), introduced into the leader of strain CM4-184 by polymerase chain reaction-mediated mutagenesis. To create CAT and F-CAT, the XhoI-ClaI fragment of Lm-CAT and LmF-CAT, respectively, was replaced by the following oligonucleotide: 5'-TCGAGATGCTTGTATTTACCCTATATACCCTAGTAACCCCTTAT-3' sense strand. Clones (LaTTG, LaTAG, LAs, LAB, LABs, Lnear a, Lmid a, Lfar a, Lst1, Lstrst1, Lst1-resto)-CAT were made by replacing the XhoI-NheI fragment of Lm-CAT with oligonucleotides containing the respective mutations (see Figs. 4, 5, and 7). Construct Lstrst2-CAT, in addition to the mutation in the XhoI-NheI region, contains mutations in the Eco0109-ClaI fragment (see Fig. 4). Constructs Lstrst2-CAT and Lnobif-CAT were made by replacing the NheI-BglII and the XhoI-SpeI fragments of Lm-CAT, respectively, with oligonucleotides containing the mutations (see Fig. 5). LFhp-CAT contains a stable stem-loop structure between sORFs A and B. This construct was made by replacing the XhoI-NheI fragment of LmF-CAT with the oligonucleotide 5'-TCGAGATGTGTGAGTAGTTCCCAGATAAGGGGCGCGTTCGCCTGCTTCAACAGTGCTTGGACGGGCAACGCGCAATTAGGGTTCTTATAGGGTTTCG-3'. The sequence in bold forms the stem-loop structure. The mini-leader was constructed by first replacing the BglII-ClaI fragment of Lm-CAT with the oligonucleotide 5'-GATCTAGAGGTAAAGCTTGTATTTACCCTATTTACCCTATTTACCCTAGTAACCCCTTAT-3' (bold nucleotides indicate introduced XbaI and HindIII sites) and then by inserting the oligonucleotide 5'-CTAGAGGTAAGACGATGGAAATTTGATAGAGGTACGTTACTATACTTATACTATACGCTAAGGGAATGCTTGTAA-3' between the XbaI and HindIII sites. In micro-leader 1, the SpeI-ClaI fragment of LmF-CAT was replaced with the oligonucleotide 5'-CTAGTGAATGGTTGTATTTACCCTATATACCCTAGTAACCCCTTAT-3'. The G in bold represents the C to G mutation introduced to create a more favorable initiation codon context for sORF F. In micro-leader 2, the NheI-SpeI fragment of micro-leader 1 was replaced with the oligonucleotide 5'-CTAGTGGGTTTCCGTCCAAGCACTGTTGAAGCAGGAAAACCCG-3'.

In Vitro Transcription and Translation-- RNA was prepared by in vitro transcription of linearized plasmids, using SP6-, T3 (Boehringer Mannheim)-, or T7 (Biofinex)-RNA polymerase according to the manufacturer's instructions. For preparation of capped transcripts, the cap analogue 7mGpppG was added to a final concentration of 0.5 mM (10 times higher than the GTP concentration). The RNA was purified by precipitation once with 3 M lithium chloride and twice with ethanol. The integrity of the synthesized transcripts was evaluated on a 6% denaturing polyacrylamide gel. RNA was quantified by measuring the absorbance at 260 nm. Equimolar amounts of transcripts (0.5 pmol) were translated in vitro in a wheat germ extract prepared according to Roberts and Paterson (27) provided with [35S]Met. In standard reactions, KAc was added to a final concentration of 100 mM. The Mg2+ concentration was 1.5 mM. The translation reaction was carried out in a final volume of 25 µl at 27 °C. After 1 h incubation, 3.5 µl of the reaction mixture was analyzed by SDS-polyacrylamide gel electrophoresis. Gels were fixed for 30 min in 50% methanol, 12% acetic acid, and then dried and exposed to x-ray films. Quantification was carried out using a PhosphorImager (Molecular Dynamics).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Internal Initiation or Shunting?-- In previous in vivo studies of the ribosome shunt mechanism, it was difficult to demonstrate conclusively that the 35 S RNA leader does not contain an IRES. The ultimate test of whether a particular sequence can direct internal initiation of translation is to introduce it between two cistrons and demonstrate that expression of the second cistron is independent of the first (28, 29). However, the poly(A) site in the leader becomes fully active if it is moved further away from the transcription start site (6). Insertion of a coding region between the transcription initiation site and the leader would therefore lead to termination of the RNA after the first cistron in vivo. The development of a cell-free translation system in which the 35 S RNA functions to direct translation (18) allows the question of whether there is any internal initiation to be directly addressed.

Translation of a reporter construct (U1A-L-CAT), in which the leader was placed between two ORFs (encoding the small nuclear ribonucleoprotein U1A and CAT, respectively), was compared with control transcripts in the wheat germ translation system. No detectable levels of CAT were produced, although U1A expression was not affected (Fig. 2). In the control construct without the upstream U1A cistron (L-CAT) but still with the leader, however, CAT was expressed with 25% efficiency compared with a short-leader construct, confirming earlier data (18). If internal initiation would have occurred, then similar levels of CAT expression would have been expected from both constructs, U1A-L-CAT and L-CAT. This result is therefore incompatible with translation by internal initiation of the type described for picornaviruses (30, 31), but it is compatible with shunting.


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Fig. 2.   CaMV 35 S RNA leader in a dicistronic construct. Constructs used for in vitro translation in a wheat germ system and the relative efficiencies of expression as determined by PhosphorImager quantification. The numbers represent the average of two independent experiments.

Scanning and/or Shunting-- To compare shunting quantitatively with linear scanning, we introduced a frameshift mutation into sORF F of plasmid Lm-CAT to create an F::CAT ORF fusion (construct LmF-CAT, Fig. 3A). In the virus context, sORF F overlaps ORF VII (Fig. 1B), and in strain S, sORF F contains an internal AUG (F'). If translation-competent ribosomes can reach the 3'-end of the leader by a scanning related process (leaky scanning, re-initiation), they encounter the two AUGs of sORF F, 91 and 64 nucleotides upstream of the AUG of ORF VII, now the AUG of CAT, producing F::CAT and F'::CAT proteins. Alternatively, translation-competent ribosomes could reach the 3'-end of the leader by shunting. Since the major shunt acceptor site is located downstream of the internal AUG codon within sORF F (17), translation initiation would occur at the start codon of CAT.


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Fig. 3.   Shunting efficiency in the wheat germ in vitro system. A, constructs used for in vitro translation. The AUG start codons of F::CAT, F'::CAT, and CAT are shown by asterisks. The arrows indicate the nonlinear migration of the shunting ribosomes, landing within sORF F. Translation products were quantified using a PhosphorImager and corrected for the methionine content. The relative percentage of initiation events at the F and CAT AUGs are indicated in each case. Numbers are the average of 10 independent experiments. B, representative in vitro translation experiment of the constructs shown in A. Positions of F, CAT, F::CAT, F'::CAT, and X proteins are indicated.

At least 80% of total translation products derived from Lm-CAT and LmF-CAT were CAT protein (Fig. 3B, lanes 1 and 2). Initiation of translation in LmF-CAT occurred also at the 5' proximal and the internal AUG codons of sORF F with a combined efficiency of ~20%, resulting in the synthesis of F::CAT and F'::CAT proteins (lane 2). The sORF F AUG is much more efficiently recognized in the short-leader constructs, CAT and FCAT (Fig. 3A). Translation of the CAT mRNA, which contains sORF F in wild-type configuration, resulted in the expression of F (Fig. 3B, lane 3), whereas translation of F-CAT mRNA, with sORF F in frameshifted configuration, resulted in the expression of F::CAT protein (lane 4). The presence of the F AUG in the short-leader RNAs still allowed initiation at the CAT AUG at a level of ~20%, probably because the suboptimal context of the F AUG (32, 33) allows some leaky scanning. Hence, the CAT/F::CAT ratio is switched from 1:4 in the short-leader RNA (lane 4) to 4:1 in the leader-containing RNA (lane 2). These results confirm that the complete leader contains elements that support shunting and that the major shunt acceptor site is located downstream of the F' start codon (17). On the other hand, they also show that despite the complexity of the leader and the presence of six other sORFs, a considerable fraction of the ribosomes reaches the sORF F and F' start codons, probably by scanning and reinitiation or by shunting employing minor shunt donor and acceptor sites.

A band migrating slower than the F::CAT fusion protein was detected upon translation of the CAT mRNA (Fig. 3, lane 3, band X). This protein was recognized by anti-CAT antibodies (not shown). Appearance of this band was only observed when significant amounts of F were produced, suggesting that it may be linked to ORF F translation, possibly via frameshifting.

To exclude the possibility that initiation at the CAT AUG in the LmF-CAT RNA was accomplished by a distance-dependent reinitiation mechanism as suggested for the yeast GCN4 RNA (34), a strong hairpin was introduced at position 86, between sORFs A and B (LhpF-CAT; Fig. 3A). Such hairpins have been shown to significantly inhibit the scanning process without interfering with shunting (17). Translation of LFhp-CAT mRNA resulted in a single protein band corresponding to initiation at the CAT AUG; no initiation event was detected at the sORF F AUGs (Fig. 3B, lane 5). This result demonstrates that the hairpin indeed inhibited linear scanning, that ribosomes that will translate F have to scan at least past position 86, and that most of those that translate CAT will have to shunt from a shunt donor located upstream of nucleotide 86.

Stem Structures Required for Shunting-- The 35 S RNA leader folds into a complex structure that can be divided into three main stem sections separated by bifurcations (Fig. 1C, see Refs. 14 and 17). Stem section 1 constitutes the most stable portion of this structure. It consists of three base paired regions each including a stretch of three G-C pairs, which are separated by bulges and followed by a bifurcation. It is striking that the shunt donor site maps to the base of stem section 1 (or just upstream of it) and the acceptor site to a region just downstream of stem section 1. Thus, stem section 1 might function in bringing the shunt donor and acceptor sites into close spatial proximity to promote shunting (Fig. 1C). To test this hypothesis, the base paired regions in stem section 1 were destroyed by introduction of 13 point mutations into the left arm (Lst1-CAT; Fig. 4). This resulted in a 90% reduction of translation. When the structure was restored with complementary mutations on the right arm (Lst1-resto-CAT), the original level of CAT protein was obtained. Thus, stem section 1 appears to be a key element for shunting. Since the wild-type construct and Lst1-resto-CAT behave similarly despite differences in their primary sequence, the structure of stem section 1 rather than its sequence is important. Introduction of the mutated stem section 1 into LmF-CAT to yield LFst1-CAT resulted in the synthesis of the F::CAT and F'::CAT fusion proteins at comparable or slightly higher levels than with the LmF leader, indicating that the scanning (or minor shunting) process was not much affected by this mutation (compare lanes 4 and 5 in Fig. 4C). However, CAT expression by shunting ribosomes was again drastically reduced.


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Fig. 4.   Importance of the 35 S RNA leader stem section 1 for ribosome shunting. A, schematic representation of the stem section 1 of the 35 S RNA leader. The two sets of mutations are indicated by arrows; those introduced in Lst1-CAT are shown by bold circles. The additional complementary mutations introduced in Lst1-resto-CAT to restore the formation of stem 1 are represented by squares. B, constructs used for in vitro translation and schematic representation of the RNA structure. The translation initiation sites of F, F', CAT, F::CAT, and F'::CAT are shown by arrows. Dotted lines indicate the region where mutations where introduced. C, results of the in vitro translation of the constructs shown in B. The figure represents one of five independent experiments that gave similar results.

Stem section 1 could be functional due to the base paired regions, the bulges, or both. Bulges might for instance be targets for interaction with proteins. To test this, the bulges were replaced by base paired regions (Lstrst1-CAT). This actually led to a slightly increased shunting efficiency (Fig. 5), showing that the bulges are not important for shunting and that the increase in base pairing in stem section 1 affects shunting positively. Furthermore, the removal of the bifurcation at the top of stem section 1 in Lnobif1-CAT also had a slight positive effect on translation efficiency (Fig. 5).


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Fig. 5.   Manipulations of base pairings in stem sections 1 and 2 and their effect on shunting efficiency. Constructs contained the CAT reporter gene fused to the complete original and mutated leaders. Only stem sections 1 and 2 of the leader are shown, and the modified regions are boxed. Relative values of in vitro translation of CAT revealed by SDS-polyacrylamide gel electrophoresis are given at the bottom of the structures.

Taken together these results show that shunting relates to stem section 1 stability, rather than to the primary sequence or the presence of bulges or the bifurcation.

To test whether the other stem sections are also required, a "mini-leader" lacking stem section 3 was produced by deletion of nucleotides 226 (224)2 to 437 (455) and a "micro leader" lacking stems 2 and 3 by deletion of nucleotides 172 (170) to 497 (515) (see Fig. 1C). In these constructs, sORF F is fused in-frame with CAT and lacks the internal F'-AUG. In both cases major translation products were CAT protein, mainly derived from shunting (Fig. 6B, lanes 2 and 3), and minor products were F::CAT, presumably derived from scanning. To show that in the micro-leader CAT translation is in fact obtained by shunting rather than leaky scanning or reinitiation, the region between 115 (113) and 171 (169) was replaced by the scanning-inhibiting hairpin (micro-leader 2). This mutation abolished translation of F::CAT, whereas translation of CAT was increased, showing that in fact shunting had occurred and that stem section 1 is necessary and sufficient for this process. These results show that the first 113 nt together with the last 96 nt of the leader are sufficient for shunting.


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Fig. 6.   Stem section 1 of the CaMV 35 S RNA leader is sufficient to promote ribosome shunting. A, constructs used for in vitro translation and schematic representation of their RNA secondary structure. The CAT, F::CAT, and F'::CAT translation initiation sites are indicated by bent arrows on the RNA structure. Curved arrows represent the nonlinear migration of ribosomes with the landing pad within sORF F. Asterisks indicate the AUG start codons of F::CAT, F'::CAT, and CAT ORFs. Lines indicate the deleted region. Dotted lines in the micro-leader 2 indicate nonviral sequences. In the micro-leaders the context of the sORF F start codon was strengthened by an AUGC to AUGG mutation. B, representative in vitro translation results. CAT, F::CAT, and F'::CAT proteins are indicated.

Although stem sections 2 and 3 are dispensable, they might have a function in the context of the whole leader. In this respect, we could show that an increase in the number of base pairs in stem section 2, reducing its folding energy from -27 to -40 kcal/mol, resulted in a 2- to 3-fold increase in reporter gene expression (Fig. 5). The strengthening of stem section 2 might stabilize the folding of an RNA structure favorable for shunting, e.g. supporting stem section 1 formation. Alternatively, it might block the transit of scanning ribosomes and thereby increase the number of complexes that follow the bypass route. In a search for cellular proteins involved in shunting, a protein from turnip, a host plant for CaMV, was identified that binds to regions within stem section 2 (35). The role of the protein is unknown, but it might function by stabilizing the shunt structure or by blocking the scanning of ribosomes in a similar manner as the stem section 2 mutation.

The Role of the Short Open Reading Frames in Shunting-- Results from in vivo experiments suggest a role for the first sORF of the leader, sORF A, in shunting (17).3 sORF A terminates six nucleotides upstream of stem section 1. sORF B, the second sORF, starts 45 (43) nucleotides downstream of sORF A and at the end of this stem section (see Fig. 1C).

A series of mutations involving sORF A were tested. Improvement of the start codon context (AUAAUGU to AUAAUGG) resulted in only slight increase of CAT expression (Fig. 7, LAs-CAT). Replacement of the start codon by UUG caused a slight reduction of the amount of CAT protein translated (LaTTG-CAT). In this construct the UUG codon is preceded by an AUA codon, and the cumulative effect of two non-AUG start codons might still allow considerable sORF A translation (36). Initiation from these non-AUG start codons and also from the original AUG codon in an unfavorable sequence context might be further enhanced by the presence of stem section 1. Downstream hairpins are known to increase the efficiency of initiation at non-AUG codons (37). To abolish completely sORF A translation, its start codon was replaced by a stop codon (Fig. 7, LaTAG-CAT). This caused a reduction of translation of the CAT ORF to ~20%.


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Fig. 7.   Importance of the sORF A region for CAT expression. Mutations introduced into the XhoI-NheI region of the Lm-CAT plasmid. The two restriction sites are indicated in bold. Dashes, same bases; dots, base deletions; letters, point mutations. sORF A is underlined. Sequences inserted in Lmid a-CAT and Lfar a-CAT are shown. The arrow shows the start of stem section 1. The efficiency of CAT translation was expressed relative to the wild-type Lm-CAT. Numbers are the average of at least six independent experiments. B and C, maps of the constructs showing the sORFs affected. The arrows in C indicate the start of stem section 1.

The position of sORF A just upstream of stem section 1 is conserved between different caulimoviruses3 and even in the less related RTBV. Increasing or decreasing the distance between sORF A and stem section 1 by deletion and insertion mutagenesis, respectively (Fig. 7, LnearA-CAT, LmidA-CAT, and LfarA-CAT), reduced CAT translation.

In summary, these experiments show that sORF A enhances shunting and that the location of sORF A with respect to stem section 1 modulates this effect.

sORF A was elongated to the stop codon of sORF B (Fig. 7, LAB-CAT) by introduction of mutations in the bulges to avoid affecting the structure of stem section 1. This resulted in a strong decrease in CAT expression. Improving the start codon context of sORFA::B did not decrease CAT expression further (Fig. 7, LABs-CAT) consistent with the idea that this AUG is efficiently recognized in the original context. The inhibition of CAT translation could be due to one or more of the following reasons: (i) 80 S ribosomes translating sORF A::B could mask the shunt donor site; (ii) translation of sORF A::B could melt an important shunt structure between the original sORFs A and B (i.e. stem section 1); and (iii) ribosomes that have translated sORF A could have increased competence for shunting and/or decreased competence for scanning. After translation of A::B in LAB-CAT they would have missed the shunt donor site. The last assumption would map the shunt donor site between sORF A and sORF B, which is in agreement with the determination by hairpin insertion analysis to a position upstream of nt 86 (Fig. 3). Considering the positive effect of sORF A on ribosome shunting, the shunt donor would map downstream of sORF A, thus, between positions 73 and 86.

To test the effect of the presence of the other sORFs on translation initiation downstream of the leader, all AUGs within the 35 S RNA leader were removed (Fig. 8, LAUG-free-CAT). As expected, CAT expression was considerably increased, probably mainly because scanning can now proceed uninhibited by interfering start codons. Nevertheless, shunting also contributed to overall translation efficiency, as can be visualized by reintroducing sORF F fused to the reporter ORF (LonlyF-CAT). In this case the sum of translation events remained constant, with ~60% starting from the sORF F AUG and ~40% at the CAT AUG. We calculate that this 40% is a combined number with a contribution of ~15% from ribosomes that reach the CAT AUG by leaky scanning and ~25% from ribosomes that reach it by shunting. Re-introduction of sORF A into the sORF-free leader to yield LonlyA-CAT reduced translation, probably because sORF A inhibits scanning. Re-introduction of sORF A into the leader containing only sORF F to yield LonlyA-F-CAT reduced translation starting from the F AUG more than that from the CAT AUG, indicating that sORF A is mainly inhibitory to scanning.


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Fig. 8.   AUG-free leader. A, mutations introduced in the wild-type leader to exclude all or most of the AUG codons. B, constructs used for in vitro translation and the PhosphorImager quantification of the bands shown in C. For the leader-containing RNAs, the level of CAT synthesized from the LAUG-free-CAT RNA was taken as 100%. For the F-CAT RNA, 100% corresponded to the total amount of protein synthesized from this RNA. Numbers represent the average of six independent experiments. C, representative in vitro translation of the constructs shown in B.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The idea of nonlinear ribosome migration to reach the start codon of an ORF was suggested several years ago (21, 22, 38) and later demonstrated for the CaMV 35 S RNA leader (16, 17). Since then, similar mechanisms of translation initiation have been proposed for other viral transcripts (RTBV (19), adenovirus (20), and BFDV (23)); however, the molecular mechanism for ribosome shunting remained undefined. It has recently been shown that shunting promoted by the CaMV 35 S RNA leader can also take place in a wheat germ cell-free system (18). In the in vitro system the effect of mutations on reporter gene expression can be interpreted solely in terms of translation, since splicing, polyadenylation, or RNA transport is not involved, and mRNA levels have been shown to be more or less constant over the 1-h incubation period (18). In addition, the in vitro system allowed us to definitively prove the absence of a true IRES in the CaMV 35 S RNA leader sequence (see Fig. 2).

Translation directed by the CaMV 35 S RNA leader in the wheat germ in vitro translation system described here and previously (18) essentially reflects the efficiencies measured in Orychophragmus and other Cruciferae protoplasts derived from CaMV host plants and confirms the conclusions concerning shunting as published (15-17). In non-host protoplast systems, e.g. derived from Daucus carotae (39) and Nicotiana tabacum (15), translation downstream of the leader was much less efficient, leading to the suggestion that effective shunting depends on specific host factors. Results obtained with the wheat germ system, however, argue against this suggestion since wheat is only very distantly related to the Cruciferae and is not a host of CaMV. However, also in this system shunting depends on as yet unknown features of the extract. Previous attempts to use wheat germ extracts for the analysis of shunt had been unsuccessful,4 and we observe some differences in extracts from different sources, indicating variability in important plant factor(s). Such differences might be exploited in future complementation assays for either testing fractions from active extracts or plant proteins which interact with the CaMV 35 S RNA leader sequence.

Only a fraction of the ribosomes that start scanning on the 35 S RNA leader follow the bypass route; the rest continue scanning and probably translate one or more of the sORFs, most of which contain AUG codons in unfavorable context. Hence, the shunt model predicts that sORFs within this leader are translated by a different mechanism (if at all) than the ORF downstream of it. This has been shown indirectly by fusion of reporter ORFs to sORFs in the center of the leader (12, 16, 17). The relative amounts of protein synthesized from the reporter genes in the middle and at the end of such leaders were used as a measure of shunting efficiency. Our system allows similar determinations but with the advantage that only a minimal mutation in the leader, the deletion of a single base within sORF F, is required. The sORF F start codon is located upstream of the proposed shunt acceptor site (17), and the frameshift mutation brings this start codon and an additional internal AUG in phase with the downstream reporter ORF. This made it possible to simultaneously assay translation initiation at the F AUGs and at the CAT AUG. Our data showed that initiation events at these start codons indeed occurred by two different mechanisms. For instance the presence of a scanning-inhibiting stem structure at position 86 completely abolished expression from the F AUGs, whereas translation from the CAT AUG was not affected. This suggests that ribosomes that will translate F have to scan at least past position 86, and those that translate CAT shunt to the acceptor region before position 86 is reached. The enhancing effect of the first sORF of the leader (sORF A) strongly indicated that shunting occurs downstream of sORF A, i.e. downstream of position 73. Consequently the shunt donor site maps to a site between positions 73 and 86 that is at the base of stem section 1 and in close spatial proximity to the shunt acceptor site. The importance of stem section 1 as a crucial element in shunting was confirmed by mutations and second site reversions of this structure, which abolished and reestablished shunting, respectively. We also showed that the base paired regions rather than the bulges of stem section 1 are important.

Stem section 1 is not only necessary but also sufficient for shunting. It can be combined with short upstream and downstream sequences to form a micro-leader, which is particularly active in shunting. This micro-leader does not contain sequences from stem section 2 that were required for shunting in transient expression experiments (17). In the latter case, dicistronic constructs had been employed with one reporter ORF in the center and another one at the end of the leader. We assume that in this case the presence of the bulky central insert destabilized the stem structure, and therefore, stem sections 1 and 2 were both required to maintain the shunting structure. That the structure of stem section 2 might contribute to shunting efficiency in the context of the whole leader was shown by modifying this element (Fig. 5).

We cannot exclude the presence of additional shunt donor/acceptor pairs within the full-length leader. In fact, the relatively strong translation from the internal AUG within sORF F (F'-AUG), present in some of our constructs, suggests such a possibility. For instance, LFst1-CAT (Fig. 4) yields much more F'::CAT than F::CAT protein, suggesting that a second shunt acceptor might reside between the F and F'-AUGs. Whether stem section 2 is responsible for this effect is presently being studied.

Translation initiation requires cooperative interactions of a number of initiation and other factors with each other, with GTP, the ribosome, tRNAMet-ini and mRNA (40-42). In discussions of initiation mechanisms, a dynamic view with main emphasis on a defined pathway of these interactions is usually proposed, defining cap binding, unwinding, scanning, AUG recognition, etc. as individual steps. Different models have been advanced for the kinetic order of these steps (11, 43-45). An alternative view gives more emphasis to the final initiation complex and its number of components and interactions, while pointing out that the assembly pathway(s) leading to this complex can be different and might be of secondary importance (46).5 This view is based on analogy of the active translation initiation complex with the active transcription initiation complex formed by several proteins binding to a core promoter and enhancer sequences (47) and also with the splicing complex formed by small nuclear RNPs binding to splice donors and acceptors. In most cases, the major specific protein-RNA interactions leading to translation initiation are the ones formed between the cap and eIF-4E, and between an AUG (preferably in good context) and the ternary complex consisting of eIF2, tRNAMet-ini and GTP; other factors contribute to RNA binding energetically but with less specificity. For internal initiation the missing eIF-4E-cap interaction is thought to be compensated by optimization of other RNA-protein interactions, e.g. between the IRES and classical and additional initiation factors and the ribosome (46).

The terms scanning and shunting in the narrow sense presume the dynamic view, i.e. after cap recognition a search is started along the RNA for the closest AUG (scanning), whereas certain regions can be skipped (shunting). In the wider sense, scanning and shunting would just reflect the strong preference for RNA components in linear or spatial vicinity, respectively, to participate in initiation complex assembly. The importance of stem section 1 in shunting and the mapping of the shunt donor and acceptor sites is in accordance with this view, since the stem structure obviously brings shunt donor and acceptor into close spatial proximity.

It is interesting to note that in the other cases where shunting has been reported, stem structures also seem to be involved, i.e. for the RTBV leader (19), the adenovirus tripartite leader (20), and the BFDV leader (23). In the latter case the stem structure is proposed to be stabilized by a virus protein.

The effect of sORF A on shunting adds a dynamic component to the shunting process, since its effect can best be explained if sORF A is translated prior to shunting. We hypothesize that after sORF A translation all of the initiation factors required for reinitiation and shunting remain attached to the ribosome, whereas factors required for scanning, and thereby unwinding secondary structural elements, might have been removed (17). Until those are recruited again, shunting may be favored over linear scanning which would require melting of stem section 1. Factors important for the reinitiation competence of the initiation complex might be, for instance, eIF-4G, 3 and 2, while eIF-4A and 4B, due to their helicase activity, might be important for structure breaking and scanning.

An alternative effect of the specific configuration of sORF A and stem section 1 could be that ribosomes stall after translating sORF A. Ribosome stalling has been described for a sORF in the leader of human cytomegalovirus (48) and may be a general feature of other translation controlling sORFs (49). Stalling would be relatively independent of the initiation efficiency and would produce an efficient block to the migration of subsequently following ribosomes. In the special case of CaMV, these following ribosomes might be induced to shunt.

At present, data obtained with sORF A mutants only show that a translational process at this sORF and the proper spatial localization of this process are important determinants of shunting efficiency, but they do not allow us to discriminate between different mechanistic models. This would require an analysis of ribosome flow in the CaMV 35 S RNA leader. Such analyses would be very difficult in vivo but are possible in the in vitro translation system. Experiments using different translation inhibitors and centrifugation methods to analyze directly ribosome association with leader RNAs containing different sORF A mutations will produce further insights into the ribosome shunt mechanism.

    ACKNOWLEDGEMENTS

We thank David Kirk for excellent technical help, Mike Rothnie for some of the graphical work, and Helen Rothnie and Manfred Heinlein for helpful comments on the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Institut für Biochemie und Molekularbiologie, Bern University, Bühlstrasse 28, 3012 Bern, Switzerland.

To whom correspondence should be addressed. Tel.: 4161-697 66 84; Fax: 4161-697 39 76; E-mail: Hohn{at}fmi.ch.

1 The abbreviations used are: CaMV, cauliflower mosaic virus; IRES, internal ribosome entry site; ORF, open reading frame; sORF, short open reading frames; RTBV, rice tungro bacilliform virus; CAT, chloramphenicol acetyltransferase; nt, nucleotide(s); eIF, eukaryotic initiation factor; BFDV, budgerigar fledling discase virus.

2 The numbering refers to our modified leader; in brackets the corresponding position in CaMV 35 S RNA leader as used by Fütterer et al. (16) is given.

3 M. M. Pooggin, J. Fütterer, and T. Hohn, manuscript in preparation.

4 K. Gordon, J. Fütterer, and T. Hohn, unpublished observations.

5 E. Wimmer, personal communication.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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