Selective expansion of the newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes

The structural variants of the regulatory and coding regions of the LTR-retrotransposon 1731 are described. Two classes of genomic copies of retrotransposon 1731, with and without frameshifting strategy to express Gag and Pol proteins, were earlier revealed in the D. melanogaster genome. Copies without frameshifting are shown to be evolved from an ancient variant with frameshifting and are widespread in the genomes of the melanogaster complex species. Position of a rare codon responsible for ribosome pausing and efficient frameshifting is identified. Two structural variants of 1731 LTRs were detected in the melanogaster complex species: the predominant structural variant A1A2 of 1731 LTR in the D. melanogaster, D. simulans, and D. sechellia genomes contains duplicated and diverged copies of 28 bp in the U3 region, whereas A1 variant lacking this duplication is expanded in the D. mauritiana genome. Selective expansion of the A1A2 variant was detected in the independently established D. melanogaster cell cultures. A1A2 variant is expressed in embryos, cell culture, and testes, whereas A1 is expressed only in testes of D. melanogaster. Relief of expression of the A1A2 but not A1 variant in the ovaries as a result of mutation in the RNA interference (RNAi) spn-E gene is shown. Thus, expansion of the recently evolved genomic variants of the LTR retrotransposon 1731 possessing a new translation strategy, duplication in the U3 region, and extended profile of expression is revealed.     

Polyamine sensing during antizyme mRNA programmed frameshifting

A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5' and a pseudoknot 3' of the shift site. The present work illustrates that the ORF1 stop codon and the 5' element are critical for polyamine sensing, whereas the 3' pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.     

Expression of the green fluorescent protein in Paramecium tetraurelia

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.     

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.     

A −1 frameshift in the HIV-1 env gene is enhanced by arginine deficiency via a hungry codon mechanism

Ribosomal frameshifting is used by various organisms to maximize protein coding potential of genomic sequences. It is commonly exploited by RNA viruses to overcome the constraint of their limited genome size. Frameshifting requires specific RNA structural features, such as a suitable heptanucleotide “slippery” sequence and an RNA pseudoknot. Previous genomic analysis of HIV-1 indicated the potential for several hidden genes encoded through frameshifting; one of these, overlapping the envelope gene, has an RNA pseudoknot just downstream from a slippery sequence, AAAAAGA that features an adenine quadruplet prior to a potential hungry arginine codon (AGA). This env-frameshift (env-fs) gene has been shown to encode a truncated glutathione peroxidase homologue, with both antioxidant and anti-apoptotic activities in transfected cells. Using a dual reporter cell-based frameshift assay, we demonstrate that the env-fs frameshift sequence is active in vitro. Furthermore, in arginine deficient media, env-fs frameshifting increased over 100% (p < 0.005), consistent with the hypothesized hungry codon mechanism. As a response to arginine deficiency, increased expression of the antioxidant viral GPx gene (env-fs) by upregulation of frameshifting could be protective to HIV-infected cells, as a countermeasure to the increased oxidative stress induced by arginine deficiency (because NO is a known scavenger of hydroxyl radical).     

Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast.

The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF. Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF. The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels. An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting. Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1. The product contains an extra amino acid corresponding to the shift site. The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe. This frameshifting is 80% +1 and 20% -2. The response of S. pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S. cerevisiae. S. pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.     

Extreme dependence of gH and gL expression on ORF57 and association with highly unusual codon usage in rhesus monkey rhadinovirus

Standard vectors for high-level expression elicited undetectable levels of the gH and gL glycoproteins of rhesus monkey rhadinovirus (RRV) following transient-transfection assays under a variety of conditions. These same vectors and conditions yielded high levels of RRV gB expression. Unlike other genes of RRV, both the gH and gL genes were noted to have a highly aberrant, suboptimal codon usage. High levels of RRV gH and gL expression were achieved by two alternative means: codon optimization or coexpression of RRV ORF57. The failure of gH and gL to be expressed in the absence of ORF57 and in the absence of codon optimization could not be explained by the failure of RNA to egress from the nucleus. Rather, the defect in gH and gL expression appeared to be cytoplasmic in nature. It is not clear at the present time whether the aberrant codon usage for gH and gL of RRV is an intentional regulatory strategy used by the virus or whether it is driven by some external force, such as intrinsic immunity. In any event, our results indicate that the need of ORF57 for gH and gL expression can be circumvented by codon optimization, that RRV ORF57 acts principally to allow translation of gH and gL RNA in the cytoplasm, and that this activity of ORF57 is related in some way to the aberrant codon usage of the gH and gL RNAs.     

Structural heterogeneity and genomic distribution of Drosophila melanogaster LTR-retrotransposons

Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, beta-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.     

Analyses of frameshifting at UUU-pyrimidine sites.

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.     

Expression levels influence ribosomal frameshifting at the tandem rare arginine codons AGG_AGG and AGA_AGA in Escherichia coli

The rare codons AGG and AGA comprise 2% and 4%, respectively, of the arginine codons of Escherichia coli K-12, and their cognate tRNAs are sparse. At tandem occurrences of either rare codon, the paucity of cognate aminoacyl tRNAs for the second codon of the pair facilitates peptidyl-tRNA shifting to the +1 frame. However, AGG_AGG and AGA_AGA are not underrepresented and occur 4 and 42 times, respectively, in E. coli genes. Searches for corresponding occurrences in other bacteria provide no strong support for the functional utilization of frameshifting at these sequences. All sequences tested in their native context showed 1.5 to 11% frameshifting when expressed from multicopy plasmids. A cassette with one of these sequences singly integrated into the chromosome in stringent cells gave 0.9% frameshifting in contrast to two- to four-times-higher values obtained from multicopy plasmids in stringent cells and eight-times-higher values in relaxed cells. Thus, +1 frameshifting efficiency at AGG_AGG and AGA_AGA is influenced by the mRNA expression level. These tandem rare codons do not occur in highly expressed mRNAs.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

A -1 frameshift in the HIV-1 env gene is enhanced by arginine deficiency via a hungry codon mechanism

Ribosomal frameshifting is used by various organisms to maximize protein coding potential of genomic sequences. It is commonly exploited by RNA viruses to overcome the constraint of their limited genome size. Frameshifting requires specific RNA structural features, such as a suitable heptanucleotide “slippery” sequence and an RNA pseudoknot. Previous genomic analysis of HIV-1 indicated the potential for several hidden genes encoded through frameshifting; one of these, overlapping the envelope gene, has an RNA pseudoknot just downstream from a slippery sequence, AAAAAGA that features an adenine quadruplet prior to a potential hungry arginine codon (AGA). This env-frameshift (env-fs) gene has been shown to encode a truncated glutathione peroxidase homologue, with both antioxidant and anti-apoptotic activities in transfected cells. Using a dual reporter cell-based frameshift assay, we demonstrate that the env-fs frameshift sequence is active in vitro. Furthermore, in arginine deficient media, env-fs frameshifting increased over 100% (p < 0.005), consistent with the hypothesized hungry codon mechanism. As a response to arginine deficiency, increased expression of the antioxidant viral GPx gene (env-fs) by upregulation of frameshifting could be protective to HIV-infected cells, as a countermeasure to the increased oxidative stress induced by arginine deficiency (because NO is a known scavenger of hydroxyl radical).     

An integrated view of the correlations between genomic and phenomic variables

Genome sequencing opened the flood gate of ＂-omics＂ studies, among which the research about correlations between genomic and phenomic variables is an important part. With the development of functional genomics and systems biology, genome-wide investigation of the correlations between many genomic and phenomic variables became possible. In this review, five genomic variables, such as evolution rate （or ＂age＂ of the gene）, the length of intron and ORF （protein length） in one gene, the biases of amino acid composition and codon usage, along with the phenomic variables related to expression patterns （level and breadth） are focused on. In most cases, genes with higher mRNA/protein expression level tend to evolve slowly, have less intronic DNA, code for smaller proteins, and have higher biases of amino acid composition and codon usage. In addition, broadly expressed proteins evolve more slowly and are shorter than tissue-specific proteins. Studies in this field are helpful for deeper understanding the signatures of selection mediated by the features of gene expression and are of great significance to enrich the evolution theory.     

Identification of Hepta- and Octo-Uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants

Programmed frameshifting is one of the translational recoding mechanisms that read the genetic code in alternative ways. This process is generally programmed by signals at defined locations in a specific mRNA. In this study, we report the identification of hepta- and octo-uridine stretches as sole signals for programmed +1 and −1 ribosomal frameshifting during translation of severe acute respiratory syndrome coronavirus (SARS-CoV) ORF 3a variants. SARS-CoV ORF 3a encodes a minor structural protein of 274 amino acids. Over the course of cloning and expression of the gene, a mixed population of clones with six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon was found. In vitro and in vivo expression of clones with six, seven and eight Ts, respectively, showed the detection of the full-length 3a protein. Mutagenesis studies led to the identification of the hepta- and octo-uridine stretches as slippery sequences for efficient frameshifting. Interestingly, no stimulatory elements were found in the sequences upstream or downstream of the slippage site. When the hepta- and octo-uridine stretches were used to replace the original slippery sequence of the SARS-CoV ORF 1a and 1b, efficient frameshift events were observed. Furthermore, the efficiencies of frameshifting mediated by the hepta- and octo-uridine stretches were not affected by mutations introduced into a downstream stem–loop structure that totally abolish the frameshift event mediated by the original slippery sequence of ORF 1a and 1b. Taken together, this study identifies the hepta- and octo-uridine stretches that function as sole elements for efficient +1 and −1 ribosomal frameshift events.     

<Emphasis Type="Italic">HB</Emphasis> mobile element in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> genome: Structural and functional analyses

The structure was analyzed for 60 annotated copies of the mobile genetic element (MGE) HB from the Drosophila melanogaster genome. The genomic distribution of HB copies was studied, and preferential insertion sites (hot spots) were identified, which presumably amount to several kilobases. Structural analysis of the open reading frame (ORF) and terminal repeats of HB was performed. All 26 HB copies retaining the ORF sequence have a stop codon in the same position. Consequently, the HB ORF proved indeed to code for an enzyme of 148 amino acid residues, relatively small for Tc1-family transposases. The ORF consensus sequence was established. HB{}1185 was identified as the only HB copy potentially coding for a functional protein. All 37 repeat-containing HB copies were analyzed. Of these, only four had functional terminal sequences, lacking, however, a functional transposase gene. A new 7762-bp copy of MGE roo was found in the D. melanogaster genome; the copy was earlier unavailable from databases and represents an insert in the HB{}1605 sequence.