Antisense Reading Frames Research Articles

The Ribosome as a Missing Link in Prebiotic Evolution III: Over-Representation of tRNA- and rRNA-Like Sequences and Plieofunctionality of Ribosome-Related Molecules Argues for the Evolution of Primitive Genomes from Ribosomal RNA Modules.

We propose that ribosomal RNA (rRNA) formed the basis of the first cellular genomes, and provide evidence from a review of relevant literature and proteonomic tests. We have proposed previously that the ribosome may represent the vestige of the first self-replicating entity in which rRNAs also functioned as genes that were transcribed into functional messenger RNAs (mRNAs) encoding ribosomal proteins. rRNAs also encoded polymerases to replicate itself and a full complement of the transfer RNAs (tRNAs) required to translate its genes. We explore here a further prediction of our “ribosome-first” theory: the ribosomal genome provided the basis for the first cellular genomes. Modern genomes should therefore contain an unexpectedly large percentage of tRNA- and rRNA-like modules derived from both sense and antisense reading frames, and these should encode non-ribosomal proteins, as well as ribosomal ones with key cell functions. Ribosomal proteins should also have been co-opted by cellular evolution to play extra-ribosomal functions. We review existing literature supporting these predictions. We provide additional, new data demonstrating that rRNA-like sequences occur at significantly higher frequencies than predicted on the basis of mRNA duplications or randomized RNA sequences. These data support our “ribosome-first” theory of cellular evolution.

Antisense-Derived HIV-1 Cryptic Epitopes Are Not Major Drivers of Viral Evolution during the Acute Phase of Infection.

While prior studies have demonstrated that CD8 T cell responses to cryptic epitopes (CE) are readily detectable during HIV-1 infection, their ability to drive escape mutations following acute infection is unknown. We predicted 66 CE in a Zambian acute infection cohort based on escape mutations occurring within or near the putatively predicted HLA-I-restricted epitopes. The CE were evaluated for CD8 T cell responses for patients with chronic and acute HIV infections. Of the 66 predicted CE, 10 were recognized in 8/32 and 4/11 patients with chronic and acute infections, respectively. The immunogenic CE were all derived from a single antisense reading frame within pol However, when these CE were tested using longitudinal study samples, CE-specific T cell responses were detected but did not consistently select for viral escape mutations. Thus, while we demonstrated that CE are immunogenic in acute infection, the immune responses to CE are not major drivers of viral escape in the initial stages of HIV infection. The latter finding may be due to either the subdominant nature of CE-specific responses, the low antigen sensitivity, or the magnitude of CE responses during acute infections.IMPORTANCE Although prior studies demonstrated that cryptic epitopes of HIV-1 induce CD8 T cell responses, evidence that targeting these epitopes drives HIV escape mutations has been substantially limited, and no studies have addressed this question following acute infection. In this comprehensive study, we utilized longitudinal viral sequencing data obtained from three separate acute infection cohorts to predict potential cryptic epitopes based on HLA-I-associated viral escape. Our data show that cryptic epitopes are immunogenic during acute infection and that many of the responses they elicit are toward translation products of HIV-1 antisense reading frames. However, despite cryptic epitope targeting, our study did not find any evidence of early CD8-mediated immune escape. Nevertheless, improving cryptic epitope-specific CD8 T cell responses may still be beneficial in both preventative and therapeutic HIV-1 vaccines.

The Novel Anaerobiosis-Responsive Overlapping Gene ano Is Overlapping Antisense to the Annotated Gene ECs2385 of Escherichia coli O157:H7 Sakai.

Current notion presumes that only one protein is encoded at a given bacterial genetic locus. However, transcription and translation of an overlapping open reading frame (ORF) of 186 bp length were discovered by RNAseq and RIBOseq experiments. This ORF is almost completely embedded in the annotated L,D-transpeptidase gene ECs2385 of Escherichia coli O157:H7 Sakai in the antisense reading frame -3. The ORF is transcribed as part of a bicistronic mRNA, which includes the annotated upstream gene ECs2384, encoding a murein lipoprotein. The transcriptional start site of the operon resides 38 bp upstream of the ECs2384 start codon and is driven by a predicted σ70 promoter, which is constitutively active under different growth conditions. The bicistronic operon contains a ρ-independent terminator just upstream of the novel gene, significantly decreasing its transcription. The novel gene can be stably expressed as an EGFP-fusion protein and a translationally arrested mutant of ano, unable to produce the protein, shows a growth advantage in competitive growth experiments compared to the wild type under anaerobiosis. Therefore, the novel antisense overlapping gene is named ano (anaerobiosis responsive overlapping gene). A phylostratigraphic analysis indicates that ano originated very recently de novo by overprinting after the Escherichia/Shigella clade separated from other enterobacteria. Therefore, ano is one of the very rare cases of overlapping genes known in the genus Escherichia.

The contribution of CD8 T cells targeting Cryptic Epitopes on viral evolution during acute HIV-1 infection.

Abstract Alternative reading frames (ARF) of HIV, in both sense and antisense directions, have been shown to produce polypeptides of unknown functions. These peptides, known as cryptic epitopes (CE), commonly induce CD8 T cell responses in HIV; however, their role in controlling HIV-1 infection remains unclear. We hypothesized that CD8 T-cells target CE shortly following acute HIV infection and force viral escape. Such a finding would indicate that CE may serve as viable immunogens for inclusion in preventative HIV-1 vaccines. Using an HLA-I binding prediction algorithm applied to fixed mutations in ARF during the first two years of infection in a Zambian patient cohort, we predicted 66 CD8 T-cell escape mutations within gag, pol, and nef, which we further tested for immunogenicity. 8/32 chronic patient samples responded to at least one predicted CE as shown by IFNγ ELISpot. Furthermore, all ten immunogenic CE identified in the study were derived from a single antisense reading frame within the coding region of pol. Longitudinal studies failed to show any evidence of early viral escape to CE, as evaluation of these CE-specific CD8 T-cells following acute infection was not temporally associated with the occurrence of potential escape mutations. Our data suggest that although T-cell responses to CE can be detected during early HIV infections, they rarely establish HIV-1 escape mutations. As such their contribution to viral control is likely limited. Potential reasons for their limitations in forcing escape mutations include their infrequent expression and the weak magnitude of T-cell responses generated. Nevertheless, these data indicate that cryptic epitopes are not optimal targets for a preventative HIV vaccine.

The Role of Dipeptide Repeats in C9ORF72-Related ALS-FTD.

Expansion of a hexanucleotide (GGGGCC) repeat in the gene chromosome 9 open reading frame 72 (C9ORF72) is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Three non-exclusive mechanisms have been proposed to contribute to the pathology initiated by this genetic insult. First, it was suggested that decreased expression of the C9orf72 protein product may contribute to disease. Second, the recognition that C9ORF72-related disease is associated with accumulation of GGGGCC repeat-containing RNA in nuclear foci led to the suggestion that toxic gain of RNA function, perhaps related to sequestration of RNA-binding proteins, might be an important driver of disease. Third, it was subsequently appreciated that GGGGCC repeat-containing RNA undergoes unconventional translation to produce unnatural dipeptide repeat (DPR) proteins that accumulate in patient brain early in disease. DPRs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DPRs: glycine–alanine (GA), glycine–arginine (GR), proline–alanine (PA), proline–arginine (PR) and glycine–proline (GP; GP is generated from both the sense and antisense reading frames). However, the relative contribution of each DPR to disease pathogenesis remains unclear. Here, we review evidence for the contribution of each specific DPR to pathogenesis and examine the probable mechanisms through which these DPRs induce neurodegeneration. We also consider the association of the toxic DPRs with impaired RNA metabolism and alterations to the liquid-like state of non-membrane-bound organelles.

Antisense transcript derived HIV-1 cryptic epitopes are recognized by T cells during chronic infection.

Abstract Alternative reading frames (ARF) of HIV, both sense and antisense directions, have shown to produce polypeptides of unknown functions. Although the roles of these polypeptides are unclear, they can still potentially act as HIV derived antigens, eliciting host T cell responses. These ARF derived epitopes, known as cryptic epitopes (CE), occur commonly in HIV, SIV, and other retroviruses through ribosomal frame-shifting, internal ribosomal entry sites, and alternative start codon initiation. We hypothesize that T cell responses to CE are capable of driving viral escape mutations at a population level. In order to identify CE of HIV capable of driving viral escape, sequence changes in gag, pol, and nef of HIV were identified during acute infection in a cohort of Zambian serodiscordant couples. HLA-I associated polymorphisms in ARF can thus be identified, predicting potentially immunogenic CE. Several CE were identified as a result (N=66), in which the majority appears to have been derived from antisense ARF. These CE were used to stimulate cryopreserved PBMC samples from HIV-1 patients in the chronic stage of infection. We found 8/32 patients responded to at least one predicted CE as shown by IFNγ ELISpot. Interestingly, all 10 immunogenic CEs found in this study were derived from a single antisense reading frame in the pol coding region. Furthermore, these CE were not only capable of eliciting CD8, but also CD4 responses. When examining alternative start codons in each reading frame, we found an enrichment of CUG in the antisense frame containing the immunogenic CE. Together, our results show HIV antisense CE can be targeted by T cells and may therefore have implications for T-cell vaccines.

Immune screening identifies novel T cell targets encoded by antisense reading frames of HIV-1.

Cytotoxic-T lymphocyte (CTL) responses to epitopes in alternative HIV reading frames have been reported. However, the extent of CTL responses to putative proteins encoded in antisense reading frames is unknown. Using sequence alignments and computational approaches, we here predict five potential antisense HIV proteins and characterize common CTL responses against them. Results suggest that antisense-derived sequences are commonly transcribed and translated and could encode functional proteins that contain important targets of anti-HIV cellular immunity.

A non-stop antisense reading frame in the grp78 gene of Neurospora crassa is homologous to the Achlya klebsiana NAD-gdh gene but is not being transcribed

A long non-stop reading frame exists on the antisense strand of the grp78 gene (cDNA and genomic DNA) of Neurospora crassa. Computer analysis revealed a strong similarity of the putative antisense protein to the 10th exon of the NAD-dependent glutamate dehydrogenase gene ( NAD-gdh) of Achlya klebsiana, which is itself located on the complementary strand of a transcribed hsc70 gene homologue. In Neurospora, no grp78 antisense mRNA was detected by Northern blot and reverse transcription-coupled polymerase chain reaction analyses, indicating that this long reading frame is not being transcribed. Hypotheses for the presence of such unexpressed non-stop reading frames are discussed.

Long non-stop reading frames on the antisense strand of heat shock protein 70 genes and prion protein (PrP) genes are conserved between species.

Several mammalian genes, including heat shock protein (Hsp70) and prion protein (PrP) genes, have been reported to have long open reading frames (ORFs) or non-stop reading frames (NRFs) in the antisense direction. A simple explanation would be that these long antisense reading frames, which are usually in the same triplet frame as the coding strand, are the fortuitous byproduct of a high overall [G+C] content with concomitant preference for G/C over A/T in the third codon position, a preference for RNY type codons (purine/any nucleotide/pyrimidine), and/or a bias against serine and leucine, the only amino acids with codons that can be read as stop codons in the antisense direction. The PrP genes and most heat shock genes with long antisense NRFs (aNRFs) are indeed relatively [G+C] rich but do not show a bias against serine and leucine. In several vertebrates investigated, at least one of the Hsp70 genes has a long antisense reading frame, and we found that some, though not all, putative stop codons in long Hsp70 antisense reading frames were due to sequencing errors. The PrP gene contains an extended antisense open reading frame in all 45 eutherian mammals tested, but not in a marsupial and in a bird. In the PrP gene, the long, protein-coding exon also harbors the antisense nonstop reading frame. In both Hsp70 and PrP genes, the putative antisense protein sequence is well conserved. Even though there is no clear evidence in Hsp70 or PrP genes for the existence of the respective antisense proteins, we speculate that such antisense proteins serve to regulate the genuine Hsp and PrP proteins under special circumstances. Alternatively, regulation might occur at the RNA level, and the antisense RNA would merely lack stop codons to prevent its rapid degradation by an mRNA quality control mechanism that is triggered by premature stop codons. We note that both Hsp and PrP are involved in physiological or pathological protein aggregation phenomena, that scrapie prions have been reported to modify the expression or localization of heat shock proteins, and that in yeast, propagation of a prion-like state (PSI+) depends on a heat shock (Hsp104) protein.

Open reading frames in the antisense strands of genes coding for glycolytic enzymes in Saccharomyces cerevisiae.

Open reading frames longer than 300 bases were observed in the antisense strands of the genes coding for the glycolytic enzymes phosphoglucose isomerase, phosphoglycerate mutase, pyruvate kinase and alcohol dehydrogenase I. The open reading frames on both strands are in codon register. It has been suggested that proteins coded in codon register by complementary DNA strands can bind to each other. Consequently, it was interesting to investigate whether the open reading frames in the antisense strands of glycolytic enzyme genes are functional. We used oligonucleotide-directed mutagenesis of the PGI1 phosphoglucose isomerase gene to introduce pairs of closely spaced base substitutions that resulted in stop codons in one strand and only silent replacements in the other. Introduction of the two stop codons into the PGI1 sense strand caused the same physiological defects as already observed for pgil deletion mutants. No detectable effects were caused by the two stop codons in the antisense strand. A deletion that removed a section from -31 bp to +109 bp of the PGI1 gene but left 83 bases of the 3' region beyond the antisense open reading frame had the same phenotype as a deletion removing both reading frames. A similar pair of deletions of the PYK1 gene and its antisense reading frame showed identical defects. Our own Northern experiments and those reported by other authors using double-stranded probes detected only one transcript for each gene. These observations indicate that the antisense reading frames are not functional. On the other hand, evidence is provided to show that the rather long reading frames in the antisense strands of these glycolytic enzyme genes could arise from the strongly selective codon usage in highly expressed yeast genes, which reduces the frequency of stop codons in the antisense strand.