Alternative Reading Frames are an Underappreciated Source of Protein Sequence Novelty.

BackgroundThe major role of enzymatic toxins that target nucleic acids in biological conflicts at all levels has become increasingly apparent thanks in large part to the advances of comparative genomics. Typically, toxins evolve rapidly hampering the identification of these proteins by sequence analysis. Here we analyze an unexpectedly widespread superfamily of toxin domains most of which possess RNase activity.ResultsThe HEPN superfamily is comprised of all α-helical domains that were first identified as being associated with DNA polymerase β-type nucleotidyltransferases in prokaryotes and animal Sacsin proteins. Using sensitive sequence and structure comparison methods, we vastly extend the HEPN superfamily by identifying numerous novel families and by detecting diverged HEPN domains in several known protein families. The new HEPN families include the RNase LS and LsoA catalytic domains, KEN domains (e.g. RNaseL and Ire1) and the RNase domains of RloC and PrrC. The majority of HEPN domains contain conserved motifs that constitute a metal-independent endoRNase active site. Some HEPN domains lacking this motif probably function as non-catalytic RNA-binding domains, such as in the case of the mannitol repressor MtlR. Our analysis shows that HEPN domains function as toxins that are shared by numerous systems implicated in intra-genomic, inter-genomic and intra-organismal conflicts across the three domains of cellular life. In prokaryotes HEPN domains are essential components of numerous toxin-antitoxin (TA) and abortive infection (Abi) systems and in addition are tightly associated with many restriction-modification (R-M) and CRISPR-Cas systems, and occasionally with other defense systems such as Pgl and Ter. We present evidence of multiple modes of action of HEPN domains in these systems, which include direct attack on viral RNAs (e.g. LsoA and RNase LS) in conjunction with other RNase domains (e.g. a novel RNase H fold domain, NamA), suicidal or dormancy-inducing attack on self RNAs (RM systems and possibly CRISPR-Cas systems), and suicidal attack coupled with direct interaction with phage components (Abi systems). These findings are compatible with the hypothesis on coupling of pathogen-targeting (immunity) and self-directed (programmed cell death and dormancy induction) responses in the evolution of robust antiviral strategies. We propose that altruistic cell suicide mediated by HEPN domains and other functionally similar RNases was essential for the evolution of kin and group selection and cell cooperation. HEPN domains were repeatedly acquired by eukaryotes and incorporated into several core functions such as endonucleolytic processing of the 5.8S-25S/28S rRNA precursor (Las1), a novel ER membrane-associated RNA degradation system (C6orf70), sensing of unprocessed transcripts at the nuclear periphery (Swt1). Multiple lines of evidence suggest that, similar to prokaryotes, HEPN proteins were recruited to antiviral, antitransposon, apoptotic systems or RNA-level response to unfolded proteins (Sacsin and KEN domains) in several groups of eukaryotes.ConclusionsExtensive sequence and structure comparisons reveal unexpectedly broad presence of the HEPN domain in an enormous variety of defense and stress response systems across the tree of life. In addition, HEPN domains have been recruited to perform essential functions, in particular in eukaryotic rRNA processing. These findings are expected to stimulate experiments that could shed light on diverse cellular processes across the three domains of life.ReviewersThis article was reviewed by Martijn Huynen, Igor Zhulin and Nick Grishin

In a previous paper, it was argued that only those coding sequences descended from oligomeric repeats (the number of bases in the oligomeric unit not being a multiple of 3) can retain sufficiently long alternative open reading frames, and that such alternative open reading frames serve as the reservoir for the sudden generation of new polypeptide chains with novel functions. It was suggested that plasmid-encoded 6-amino hexanoic acid linear oligomer hydrolase that suddenly endowed Flavobacterium sp. K172 with the capacity to live off nylon by-products arose by the above mechanism. A corollary to the above argument is the expectation that those viral base sequences that are known to use two of the three alternative reading frames to encode two different polypeptide chains should invariably contain recognizable remains of the oligomeric tandem repeats, and as a consequence, various oligopeptidic repeats should also be present in the amino acid sequence of each. Furthermore, two polypeptide chains encoded by the same base sequence translated in different reading frames should show segmental homology of the type depicted previously. In the present paper, the base sequence of human hepatitis B virus ayw subtype that encodes an 832 amino acid residue long putative nucleic acid polymerase in one reading frame and a 226 residue long human hepatitis B surface antigen in the other reading frame was examined. All three predictions noted above were satisfied.

Colorectal cancer (CRC) is the third leading cause of cancer‐related deaths worldwide, affecting more than one hundred thousand men and women in the United States every year. With the incidence of early‐onset (<50 years old) CRC increasing at an alarming rate, there is an urgent and unmet need for early biomarker discovery and the identification of novel therapeutic targets in CRC. Chimeric RNAs and their protein products are well‐established as ideal biomarkers and drug targets for malignant cancers, including BCR‐ABL in chronic myelogenous leukemia and EML4‐ALK in lung cancer. Using RNA‐seq data from The Cancer Genome Atlas (TCGA) database, we have identified a chimeric RNA, denoted as A‐B, present in 45.2% of CRC samples and just 17.6% of adjacent normal margin tissue, but absent in non‐cancer colon tissues. We determined that this chimeric RNA results from cis‐splicing between adjacent genes (cis‐SAGe), and includes a splice variant of the 5’ gene, altering the reading frame of the 3’ gene. This chimeric RNA encodes a novel transmembrane protein, with the alternate reading frame on the extracellular surface, making it an ideal target for antibody therapeutics. After generating an antibody to the chimeric protein and validating its use in immunoblotting, immunoprecipitation, and immunohistochemistry, we found this novel protein is present in patient CRC samples in a distinct, punctate pattern. Overexpression of this chimeric RNA resulted in increased cell proliferation, while siRNA mediated knockdown led to a reduction in cell proliferation. We are currently investigating the function of this chimeric RNA and its biomarker potential in clinical CRC samples.

The standard genetic code (SGC) is the set of rules by which genetic information is translated into proteins, from codons, i.e. triplets of nucleotides, to amino acids. The questions about the origin and the main factor responsible for the present structure of the code are still under a hot debate. Various methodologies have been used to study the features of the code and assess the level of its potential optimality. Here, we introduced a new general approach to evaluate the quality of the genetic code structure. This methodology comes from graph theory and allows us to describe new properties of the genetic code in terms of conductance. This parameter measures the robustness of codon groups against the potential changes in translation of the protein-coding sequences generated by single nucleotide substitutions. We described the genetic code as a partition of an undirected and unweighted graph, which makes the model general and universal. Using this approach, we showed that the structure of the genetic code is a solution to the graph clustering problem. We presented and discussed the structure of the codes that are optimal according to the conductance. Despite the fact that the standard genetic code is far from being optimal according to the conductance, its structure is characterised by many codon groups reaching the minimum conductance for their size. The SGC represents most likely a local minimum in terms of errors occurring in protein-coding sequences and their translation.

Biology's ‘dark matter’ hints at fourth domain of life

Editorial overview: New protein production tools for structural biology

A structural framework for replication origin opening by AAA+ initiation factors

For nearly 80 years since the discovery of the first virus by the Russian scientist D.I. Ivanovsky, it has been recognized that all organisms of Earth's biosphere serve as natural hosts for viruses. Viruses, grouped within the informal domain Vira, infect all three domains of cellular life: archaea - Archaea, bacteria - Bacteria, and eukaryotes - Eucarya (algae, fungi, protozoa, plants, invertebrates, and vertebrates). The formation of viral population gene pools through interactions with the gene pools of their hosts has taken place under changing environmental conditions over 3.5 billion years, giving rise to the vast diversity of the virosphere. The accumulation of data on the Earth's virosphere, facilitated by the advent of high-throughput sequencing technologies (NGS), has necessitated a reassessment of approaches to virus classification and, since 2018, has led to a reform of viral taxonomy through the introduction of higher taxonomic ranks (megataxonomy). As of September 2025, the International Committee on Taxonomy of Viruses (ICTV) recognizes 15 taxonomic ranks for viruses, the most significant being: realm - 7, kingdom - 11, phylum - 23, class - 49, order - 93, family - 368, genus - 3769, and species - 16,215. Ongoing advances in metagenomics, metatranscriptomics, and the global ecology of the virosphere will inevitably drive further changes in viral taxonomy and megataxonomy. These developments are of fundamental importance for understanding the evolution of the biosphere and of practical relevance for developing new strategies to strengthen biological security and to mitigate the consequences of epidemic emergencies associated with emerging and reemerging infections.

The ribonuclease A family of proteins is well studied from the biochemical and biophysical points of view, but its evolutionary origins are obscure, as no sequences homologous to this family have been reported outside of vertebrates. Recently, the spatial structure of the ribonuclease domain from a bacterial polymorphic toxin was shown to be closely similar to the structure of vertebrate ribonuclease A. The absence of sequence similarity between the two structures prompted a speculation of convergent evolution of bacterial and vertebrate ribonuclease A-like enzymes. We show that bacterial and homologous archaeal polymorphic toxin ribonucleases with a known or predicted ribonuclease A-like fold are distant homologs of the ribonucleases from the EndoU family, found in all domains of cellular life and in viruses. We also detected a homolog of vertebrate ribonucleases A in the transcriptome assembly of the sea urchin Mesocentrotus franciscanus. These observations argue for the common ancestry of prokaryotic ribonuclease A-like and ubiquitous EndoU-like ribonucleases, and suggest a better-grounded scenario for the origin of animal ribonucleases A, which could have emerged in the deuterostome lineage, either by an extensive modification of a copy of an EndoU gene, or, more likely, by a horizontal acquisition of a prokaryotic immunity-mediating ribonuclease gene.

PCR amplification and DNA sequencing of SRV-2 from archived tumor tissues

The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus.

The gene for Kunitz peptidase inhibitor-like protein (KPILP) contains nested alternative open reading frame (aORF) that controls expression of the maternal mRNA. The content of NbKPILP mRNA in intact leaves of Nicotiana benthamiana plant is low but increases significantly upon extended dark exposure or when foreign nucleic acid is overexpressed in the cells. The NbKPILP gene promoter along with the expressed nested aORF are likely to play an important role in maintaining the levels of NbKPILP mRNA. To elucidate the role of NbKPILP promoter, we isolated a fragment of N. benthamiana chromosomal DNA upstream of the NbKPILP transcription start, sequenced it, and created constructs in which reporter E. coli uidA gene coding for β-D-glucuronidase (GUS) was placed under control of the NbKPILP promoter. By assessing the efficacy of uidA mRNA synthesis directed by the NbKPILP promoter and 35S promoter of the cauliflower mosaic virus in a transient expression system, we showed that the levels of GUS accumulation were comparable for both promoters. Prolonged incubation of the agroinjected plants in the darkness stimulated accumulation of the uidA mRNA directed by the NbKPILP promoter. Our experiments indicate that along with regulation at the transcriptional level, expression of NbKPILP mRNA can be affected by expression of the nested aORF controlled by the polypurine block (PPB) located upstream of its start codon, since introduction of mutations in the PPB resulted in significant accumulation of the NbKPILP mRNA. Nucleotide replacement in the aORF start codon led to the drastic increase in the amounts of NbKPILP mRNA and its protein product.

Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5′-CCSGG-3′ (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.

During their long evolutionary history viruses generated many proteins de novo by a mechanism called “overprinting”. Overprinting is a process in which critical nucleotide substitutions in a pre-existing gene can induce the expression of a novel protein by translation of an alternative open reading frame (ORF). Overlapping genes represent an intriguing example of adaptive conflict, because they simultaneously encode two proteins whose freedom to change is constrained by each other. However, overlapping genes are also a source of genetic novelties, as the constraints under which alternative ORFs evolve can give rise to proteins with unusual sequence properties, most importantly the potential for novel functions. Starting with the discovery of overlapping genes in phages infecting Escherichia coli, this review covers a range of studies dealing with detection of overlapping genes in small eukaryotic viruses (genomic length below 30 kb) and recognition of their critical role in the evolution of pathogenicity. Origin of overlapping genes, what factors favor their birth and retention, and how they manage their inherent adaptive conflict are extensively reviewed. Special attention is paid to the assembly of overlapping genes into ad hoc databases, suitable for future studies, and to the development of statistical methods for exploring viral genome sequences in search of undiscovered overlaps.

A model of hole transfer in DNA molecules has been proposed, which takes into account changes in the reorganization energy and orbital coupling between the neighboring bases during the charge transfer in different molecular sequences. It is shown that the rate of hole transfer by the superexchange and hopping transfer mechanisms is limited by the relaxation of the geometries of nucleobases participating in charge migration and the dynamics of solvent molecules. The rate of charge transfer in the DNA molecule is found to be dependent on the height of the potential barriers between the nucleotide and the molecular sequences. The inclusion of the interchain charge transfer, which is characterized by weak coupling between the nucleotides located in opposite strands, does not affect the general charge transport in DNA. The increase in the number of the parallel components of the hopping mechanism leads to a rise in the charge transfer rate in the double helix.