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.     

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.     

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.     

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.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

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 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.     

Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains

In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and the env/nef 3' splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3' splice site used to generate tat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3' splice site, which is the most downstream of the three rev 3' splice sites, also serves as an element inhibiting splicing at the env/nef 3' splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a different rev 3' splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3' splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev 3' splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3' splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

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。     

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5′ proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3′ hepatic control region, derived from a region ∼18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3′ splice acceptor sites causing deletion of cloned 5′ untranslated mRNA sequences and, in some cases, deletion of the 5′ end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3′ splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1–exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp. strain KKS102 was determined. Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems. When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected. The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity. These data clearly suggest that ORF2 encodes ferredoxin reductase. The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown.     

A functional splice site in the 5'' untranslated region of a zein gene.

Zein genes, the genes coding for the zein storage proteins of maize, have a unique gene structure where at least two promoters lie upstream of the coding region. Between the P1 promoter (900 base pairs upstream of the coding region) and the translation initiation AUG codon are 18 short reading frames. A discrepancy between the signals obtained by S1-mapping and primer extension and the DNA sequence in the region of one of these signals suggests the presence of a 3' splice site lying 40 nucleotides upstream of the coding region. A splicing event removing all of the short reading frames from the mRNA transcribed from the P1 promoter would bring this mRNA into a translatable form. Further evidence for a functional 3' splice site has been obtained from sequencing of primer extension products and in vitro splicing of a hybrid intron in the HeLa cell in vitro splicing system.