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.     

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.     

Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs.     

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.     

Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.     

Proteomics-driven identification of short open reading frame-encoded peptides

Accumulating evidence has shown that a large number of short open reading frames (sORFs) also have the ability to encode proteins. The discovery of sORFs opens up a new research area, leading to the identification and functional study of sORF encoded peptides (SEPs) at the omics level. Besides bioinformatics prediction and ribosomal profiling, mass spectrometry (MS) has become a significant tool as it directly detects the sequence of SEPs. Though MS-based proteomics methods have proved to be effective for qualitative and quantitative analysis of SEPs, the detection of SEPs is still a great challenge due to their low abundance and short sequence. To illustrate the progress in method development, we described and discussed the main steps of large-scale proteomics identification of SEPs, including SEP extraction and enrichment, MS detection, data processing and quality control, quantification, and function prediction and validation methods.     

Overlapping signals for translational regulation and packaging of influenza A virus segment 2

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.     

Cross-species functionality of pararetroviral elements driving ribosome shunting

Background

Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5′-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.

Methodology/Principal Findings

Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.

Conclusions/Significance

Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism.We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.     

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.     

植物小开放阅读框编码肽的研究进展

小开放阅读框（small open reading frame,sORF）一般指基因组中能够编码长度在100个氨基酸左右或以内短肽的开放阅读框。它们广泛存在于植物基因组,却因编码短肽而常被基因组注释忽视。随着翻译组学和蛋白质组学测序技术的发展,具有翻译活性的sORF被证实广泛存在于植物基因组,且参与植物生长发育等重要过程的调控。该文归纳了近些年来植物领域sORF的一些研究进展,主要包括sORF的来源与分类、信息学预测方法和生物学功能等,并基于此对植物sORF未来的研究方向进行了展望。     

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.     

The leading sequence of caulimovirus large RNA can be folded into a large stem-loop structure.

The 600 nt long sequences preceeding the first large ORFs (ORF VII) of three caulimoviruses, although varying in primary sequence, can be folded into a large stem/loop structure centered around a conserved stretch of 36 nucleotides. Deletions of the conserved sequence delay symptom appearance considerably, but do not affect expression of a reporter gene in plant protoplasts. Another striking similarity between the leaders concerns the number and distribution of small open reading frames (sORF) they carry. Expression of two of these sORFs was tested by fusion of a reporter gene: both were expressed in plant protoplasts.     

Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA.

We have studied the influence of the 600 nt long leader sequence of cauliflower mosaic virus 35S RNA on downstream translation. Plant protoplasts were transfected with plasmids expressing a CAT reporter gene from a mRNA, containing wild-type or mutant forms of the 35S RNA leader. Deletion analysis revealed the presence of three separate stimulatory sequence regions, S1, S2 and S3. The latter two interact with each other to enhance downstream translation 5- to 10-fold. This enhancement was not observed in protoplasts from a non-host plant. In the absence of either S2 or S3, the region I2, located in between, exerts an inhibitory effect on downstream translation, probably due to the presence of short open reading frames. Expression of a reporter gene inserted into I2 increases 2-fold upon deletion of either S2 or S3. We propose that mRNA regions S2 and S3 form a complex with cellular factors that allows scanning ribosomes to bypass region I2.     

Effects of alterations in the leader sequence of Rous sarcoma virus RNA on initiation of translation.

The 372-nucleotide leader sequence of Rous sarcoma virus RNA contains three conserved short open reading frames and other sequences responsible for a variety of life cycle functions. We have investigated several aspects of the leader RNA which may influence the translation of the major coding regions to which the leader is juxtaposed. We found that small perturbations of the leader length do not affect the binding and scanning of ribosomal subunits by more than about 10%, that the length and/or structure of the RSV RNA leader is near optimal for translation of the major coding regions of the viral RNA, that inclusion or deletion of open reading frames influences downstream initiation in a manner that is not strictly additive, and that reinitiation of translation at the gag gene is very efficient.     

微生物小开放阅读框:小蛋白及翻译调控研究进展

小开放阅读框(small open reading frame, sORF)广泛存在于不同生物基因组中,由于其序列短,以及编码的产物小蛋白(smallprotein,或称微蛋白;microprotein或迷你蛋白miniprotein)检测困难等原因,小开放阅读框长期未得到充分注释和研究。近年来,随着高通量测序、翻译组和质谱分析等技术的不断发展,在不同生物中发现大量新的小开放阅读框,其编码的小蛋白及介导的翻译调控已应用于药物开发及植物抗病机理等研究。但是,目前对微生物的小开放阅读框相关研究和应用还相对有限。本文综述了小开放阅读框编码产物小蛋白的发现和鉴定,以及上游开放阅读框(upstream open reading frame, uORF)对mRNA翻译调控等最新研究进展,重点介绍了微生物基因组中小开放阅读框的鉴定和功能研究进展,为深入认识微生物中小开放阅读框的功能和作用机制,以及植物和动物等高等其他生物的小蛋白和翻译调控相关研究提供参考。     

A Human Short Open Reading Frame (sORF)-encoded Polypeptide That Stimulates DNA End Joining

The recent discovery of numerous human short open reading frame (sORF)-encoded polypeptides (SEPs) has raised important questions about the functional roles of these molecules in cells. Here, we show that a 69-amino acid SEP, MRI-2, physically interacts with the Ku heterodimer to stimulate DNA double-strand break ligation via nonhomologous end joining. The characterization of MRI-2 suggests that this SEP may participate in DNA repair and underscores the potential of SEPs to serve important biological functions in mammalian cells.