Ribosome Shunting Research Articles

Ribosome shunt is essential for infectivity of cauliflower mosaic virus.

Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5'-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip plants. First- or second-site reversions appeared in the viral progeny. The second-site reversions restored shuntdependent expression to an extent correlating with their relative abundance in the progeny. Mutations that abolished both the basal and TAV-activated components of shunting proved to be lethal. Finally, by using an artificial stem structure that blocks scanning, we obtained direct evidence that ribosome shunt operates during CaMV infection.

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

The pathways of scanning ribosome migration controlled by the cauliflower mosaic virus 35 S RNA leader were investigated in vitro and in vivo. This long (600 nucleotides) leader contains several short open reading frames (sORFs) and folds into an extended hairpin structure with three main stable stem sections. Translation initiation downstream of the leader is cap-dependent and occurs via ribosomal shunt under the control of two cis elements, a short open reading frame A (sORF A) followed by stem section 1. Here we show that a second similar configuration comprising sORF B followed by stem section 2 also allows shunting. The efficiency of the secondary shunt was greatly increased when stem section 1 was destabilized. In addition, we present evidence that a significant fraction of reinitiation-competent ribosomes that escape both shunt events migrate linearly via the structured central region but are intercepted by internal AUG start codons. Thus, expression downstream of the 35 S RNA leader is largely controlled by its multiple sORFs.

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

Shunting is a translation strategy used by plant pararetroviruses (Caulimoviridae)

In eukaryotes standard initiation of translation involved 40S ribosome scanning to bridge the distance from the cap to the initiation codon. Recently deviations from that rule had been described, including “internal initiation”, “poly-A dependent translation”, and “ribosome shunting”. In ribosome shunting, ribosomes start scanning at the cap but large portions of the leader are skipped. Thereby the secondary structure of the shunted region is preserved. Scanning in plant caulimoviruses involve a small open reading frame properly spaced in front of a strong stem structure, and, in order to function, the small open reading frome has to be translated and the peptide released. This arrangement can be mimicked by artificial small open reading frames and stem structures. Shunting with viral and synthetic leaders occurs not only in plant-, but also in mammalian and yeast systems. Thus it responds to an intrinsic property of the eukaryotic translational machinery and probably acts in many cases where coding regions are preceded by complex leaders.

Role of a Short Open Reading Frame in Ribosome Shunt on the Cauliflower Mosaic Virus RNA Leader

The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5' end of the leader to a landing site near its 3' end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF.

Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems

The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning-reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5'-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5'-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of reinitiation.

Downstream Ribosomal Entry for Translation of Coronavirus TGEV Gene 3b

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5′-terminal on mRNA 3–1 and is presumably translated following 5′ cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3–1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA

Translation initiation on eukaryotic mRNAs involves 40S ribosome association with mRNA caps (m(7)GpppN), mediated by initiation factor eIF4F. 40S eukaryotic ribosomes and initiation factors undergo 5' scanning to the initiation codon, with no known role for complementarity between eukaryotic 18S rRNA and the 5' noncoding region of mRNAs. We demonstrate that the 5' noncoding region of human adenovirus late mRNAs, known as the tripartite leader, utilizes a striking complementarity to 18S rRNA to facilitate a novel form of translation initiation referred to as ribosome shunting, in which 40S ribosomes bind the cap and bypass large segments of the mRNA to reach the initiation codon. Related elements are also shown to promote ribosome shunting in adenovirus IVa2 intermediate phase mRNA during virus infection and in human heat shock protein 70 (hsp70) mRNA for selective translation during heat shock. The importance of mRNA complementarity to 18S rRNA suggests that ribosome shunting may involve either specific RNA structural features or a prokaryotic-like interaction between mRNA and rRNA.

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

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5'-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3'-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5'- and 3'-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses.

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.

Sendai virus Y proteins are initiated by a ribosomal shunt.

The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C', C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C' to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5' cap and then scan ca. 50 nucleotides (nt) of the 5' untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

Forced evolution reveals the importance of short open reading frame A and secondary structure in the cauliflower mosaic virus 35S RNA leader.

Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.

Translational Gymnastics on the Sendai Virus P/C mRNA

The Sendai virus (SeV) P/C mRNA expresses eight different polypeptide chains using a combination of ribosomal choice and cotranscriptional editing (an internal open reading frame (ORF) is accessed by the addition of a single G residue after a short run of Gs at position 1053 on the mRNA). The longest ORF within the mRNA starts at ATG104 (the second initiation site) and encodes the 568-aa P protein, an essential viral structural protein which serves both as a cofactor for the RNA-dependant RNA polymerase (L protein) and as a part of the assembly complex. The first (ACG81), third (ATG114), fourth (ATG183) and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides which are in the +1 ORF relative to P, namely C′, C, Y1, and Y2, respectively (collectively named the C proteins). Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing the C′ ACG to an ATG (GCCATG81G; ATG81/C′) ablates all expression from the downstream ATG104/P and ATG114/C initiation codons, whereas initiation from ATG183/Y1 and ATG201/Y2 remains normal in this background. Initiation from ATG183/Y1/ ATG201/Y2 probably takes place by discontinuous scanning via a ribosomal shunt. Scanning complexes appear to assemble at the 5′ cap and then scan the first ≈30 nt of the 5′ UTR before being translocated to an acceptor site close to the Y initiation codons. No specific 5′ UTR or donor site sequence elements are required, and translation of the Y proteins continues even when their start codons are changed to ACG.

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

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.

Alternative structures of the cauliflower mosaic virus 35 s RNA leader: implications for viral expression and replication

The CaMV 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600 nts leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and probably also packaging. The structure of the leader has been characterized theoretically and experimentally. The predicted conformation, a low-energy elongated hairpin, base-pairing the two halves of the leader, with a cross-like structure at the top, is strongly supported by enzymatic probing, chemical modification, and phylogenetic comparison. The elongated hairpin is stabilized by strong base-pairing between the ends of the leader, regions which are important in allowing translation downstream of the leader via the ribosome shunt mechanism. At high ionic strength the 35 S RNA leader exhibits additional higher order structures of low electrophoretic mobility: (1) a long-range pseudoknot connecting central and terminal parts of the leader; (2) a dimer. Alternative structures of the CaMV 35 S RNA leader may co-exist and have specialized functions. Their potential impact on CaMV life cycle regulation is discussed.

Translation in plants--rules and exceptions.

Translation processes in plants are very similar to those in other eukaryotic organisms and can in general be explained with the scanning model. Particularly among plant viruses, unconventional mRNAs are frequent, which use modulated translation processes for their expression: leaky scanning, translational stop codon readthrough or frameshifting, and transactivation by virus-encoded proteins are used to translate polycistronic mRNAs; leader and trailer sequences confer (cap-independent) efficient ribosome binding, usually in an end-dependent mechanism, but true internal ribosome entry may occur as well; in a ribosome shunt, sequences within an RNA can be bypassed by scanning ribosomes. Translation in plant cells is regulated under conditions of stress and during development, but the underlying molecular mechanisms have not yet been determined. Only a small number of plant mRNAs, whose structure suggests that they might require some unusual translation mechanisms, have been described.

Selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells.

Translation initiation on eukaryotic mRNAs usually occurs by 5'-processive scanning of 40S ribosome subunits from the m7GTP-cap to the initiating AUG. In contrast, picornavirus and some specialized mRNAS initiate translation by internally binding ribosomes. A poorly described third mechanism of initiation, referred to as ribosome shunting or jumping, involves discontinuous scanning by 40S ribosome subunits, in which large segments of the 5' noncoding region are bypassed. Ribosome shunting has only been observed to date on a cauliflower mosaic virus mRNA. In this report we show that the family of adenovirus late mRNAs, which are preferentially translated during infection, use a ribosome jumping mechanism to initiate protein synthesis. Late adenovirus mRNAs contain a common 5'-noncoding region known as the tripartite leader, which confers preferential translation by reducing the requirement for the rate-limiting initiation factor eIF-4F (cap-binding protein complex). Adenovirus inhibits cell protein synthesis largely by inactivating eIF-4F. We show that the tripartite leader directs both 5' linear ribosome scanning and ribosome jumping when eIF-4F is abundant but exclusively uses a ribosome jumping mechanism during late adenovirus infection or heat shock (stress) of mammalian cells, when eIF-4F is altered or inactivated. Shunting is directed by a complex group of secondary structures in the tripartite leader and is facilitated by one or more unidentified viral late gene products. We propose that shunting may represent a widespread mechanism to facilitate selective translation of specialized classes of capped mRNAs, including some stress and developmentally regulated mRNAs, which possess little requirement for eIF-4F but do not initiate by internal ribosome binding.

Position-dependent ATT initiation during plant pararetrovirus rice tungro bacilliform virus translation.

The expression of the rice tungro bacilliform virus open reading frame I was studied in transiently transfected protoplasts. Expression occurs despite the presence of a long leader sequence and the absence of a proper ATG initiation codon. Translation is initiated at an ATT codon. The efficiency of initiation in rice protoplasts depends strongly on the mechanism by which ribosomes reach this codon. From the effects of scanning-inhibiting structures inserted into different leader regions, it can be deduced that this mechanism is related to the ribosome shunt described for cauliflower mosaic virus 35S RNA. The process delivers initiation-competent ribosomes to the region downstream of the leader and is so precise that only the second of two potential start codons only 12 nucleotides apart is recognized. The ATT codon that is used when it is present downstream of the leader is hardly recognized as a start codon by ribosomes that reach it by scanning.

Picornavirus RNA translation continues to surprise

In eukaryotes standard initiation of translation involved 40S ribosome scanning to bridge the distance from the cap to the initiation codon. Recently deviations from that rule had been described, including “internal initiation”, “poly-A dependent translation”, and “ribosome shunting”. In ribosome shunting, ribosomes start scanning at the cap but large portions of the leader are skipped. Thereby the secondary structure of the shunted region is preserved. Scanning in plant caulimoviruses involve a small open reading frame properly spaced in front of a strong stem structure, and, in order to function, the small open reading frome has to be translated and the peptide released. This arrangement can be mimicked by artificial small open reading frames and stem structures. Shunting with viral and synthetic leaders occurs not only in plant-, but also in mammalian and yeast systems. Thus it responds to an intrinsic property of the eukaryotic translational machinery and probably acts in many cases where coding regions are preceded by complex leaders.