Comprehensive survey of conserved RNA secondary structures in full-genome alignment of Hepatitis C virus.

Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large 'cloud' of RNA genomes (quasispecies) which-by trial and error-comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements. Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies. Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches of Hepacivirus C are provided in the supplement.

Development of a single-chain variable antibody fragment against a conserved region of the SARS-CoV-2 spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prolonged the duration of the pandemic because of the continuous emergence of new variant strains. The emergence of these mutant strains makes it difficult to detect the virus with the existing antibodies; thus, the development of novel antibodies that can target both the variants as well as the original strain is necessary. In this study, we generated a high-affinity monoclonal antibody (5G2) against the highly conserved region of the SARS-CoV-2 spike protein to detect the protein variants. Moreover, we generated its single-chain variable antibody fragment (sc5G2). The sc5G2 expressed in mammalian and bacterial cells detected the spike protein of the original SARS-CoV-2 and variant strains. The resulting sc5G2 will be a useful tool to detect the original SARS-CoV-2 and variant strains.

Comparative Bioinformatic Analysis Reveals Conserved Regions in SARS-CoV-2 Genome for RAPID Pandemic Response.

In the face of the SARS-CoV-2 pandemic, characterized by the virus's rapid mutation rates, developing timely and targeted therapeutic and diagnostic interventions presents a significant challenge. This study utilizes bioinformatic analyses to pinpoint conserved genomic regions within SARS-CoV-2, offering a strategic advantage in the fight against this and future pathogens. Our approach has enabled the creation of a diagnostic assay that is not only rapid, reliable, and cost-effective but also possesses a remarkable capacity to detect a wide array of current and prospective variants with unmatched precision. The significance of our findings lies in the demonstration that focusing on these conserved genomic sequences can significantly enhance our preparedness for and response to emerging infectious diseases. By providing a blueprint for the development of versatile diagnostic tools and therapeutics, this research paves the way for a more effective global pandemic response strategy.

Enhancing Machine-Learning Prediction of Enzyme Catalytic Temperature Optima through Amino Acid Conservation Analysis.

Enzymes play a crucial role in various industrial production and pharmaceutical developments, serving as catalysts for numerous biochemical reactions. Determining the optimal catalytic temperature (Topt) of enzymes is crucial for optimizing reaction conditions, enhancing catalytic efficiency, and accelerating the industrial processes. However, due to the limited availability of experimentally determined Topt data and the insufficient accuracy of existing computational methods in predicting Topt, there is an urgent need for a computational approach to predict the Topt values of enzymes accurately. In this study, using phosphatase (EC 3.1.3.X) as an example, we constructed a machine learning model utilizing amino acid frequency and protein molecular weight information as features and employing the K-nearest neighbors regression algorithm to predict the Topt of enzymes. Usually, when conducting engineering for enzyme thermostability, researchers tend not to modify conserved amino acids. Therefore, we utilized this machine learning model to predict the Topt of phosphatase sequences after removing conserved amino acids. We found that the predictive model's mean coefficient of determination (R2) value increased from 0.599 to 0.755 compared to the model based on the complete sequences. Subsequently, experimental validation on 10 phosphatase enzymes with undetermined optimal catalytic temperatures shows that the predicted values of most phosphatase enzymes based on the sequence without conservative amino acids are closer to the experimental optimal catalytic temperature values. This study lays the foundation for the rapid selection of enzymes suitable for industrial conditions.

Comparative genomics of Giardia duodenalis sub-assemblage AI beaver (Be-2) and human (WB-C6) strains show remarkable homozygosity, sequence similarity, and conservation of VSP genes.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

Exploring the functionality and conservation of Alba proteins in Trypanosoma cruzi: A focus on biological diversity and RNA binding ability.

Trypanosoma cruzi is the causative agent of Chagas disease, as well as a trypanosomatid parasite with a complex biological cycle that requires precise mechanisms for regulating gene expression. In Trypanosomatidae, gene regulation occurs mainly at the mRNA level through the recognition of cis elements by RNA-binding proteins (RBPs). Alba family members are ubiquitous DNA/RNA-binding proteins with representatives in trypanosomatid parasites functionally related to gene expression regulation. Although T. cruzi possesses two groups of Alba proteins (Alba1/2 and Alba30/40), their functional role remains poorly understood. Thus, herein, a characterization of T. cruzi Alba (TcAlba) proteins was undertaken. Physicochemical, structural, and phylogenetic analysis of TcAlba showed features compatible with RBPs, such as hydrophilicity, RBP domains/motifs, and evolutionary conservation of the Alba-domain, mainly regarding other trypanosomatid Alba. However, in silico RNA interaction analysis of T. cruzi Alba proteins showed that TcAlba30/40 proteins, but not TcAlba1/2, would directly interact with the assayed RNA molecules, suggesting that these two groups of TcAlba proteins have different targets. Given the marked differences existing between both T. cruzi Alba groups (TcAlba1/2 and TcAlba30/40), regarding sequence divergence, RNA binding potential, and life-cycle expression patterns, we suggest that they would be involved in different biological processes.

Evolutionary Structure Conservation and Covariance Scores.

Effective homology search for non-coding RNAs is frequently not possible via sequence similarity alone. Current methods leverage evolutionary information like structure conservation or covariance scores to identify homologs in organisms that are phylogenetically more distant. In this chapter, we introduce the theoretical background of evolutionary structure conservation and covariance score, and we show hands-on how current methods in the field are applied on example datasets.

An ultraconserved element probe set for velvet worms (Onychophora).

Onychophora are cryptic, soil-dwelling invertebrates known for their biogeographic affinities, diversity of reproductive modes, close phylogenetic relationship to arthropods, and peculiar prey capture mechanism. The 216 valid species of Onychophora are grouped into two families - Peripatopsidae and Peripatidae - and apart from a few relationships among major lineages within these two families, a stable phylogenetic backbone for the phylum has yet to be resolved. This has hindered our understanding of onychophoran biogeographic patterns, evolutionary history, and systematics. Neopatida, the Neotropical clade of peripatids, has proved particularly difficult, with recalcitrant nodes and low resolution, potentially due to rapid radiation of the group during the Cretaceous. Previous studies have had to compromise between number of loci and number of taxa due to limitations of Sanger sequencing and phylotranscriptomics, respectively. Additionally, aspects of their genome size and structure have made molecular phylogenetics difficult and data matrices have been affected by missing data. To address these issues, we leveraged recent, published transcriptomes and the first high quality genome for the phylum and designed a high affinity ultraconserved element (UCE) probe set for Onychophora. This new probe set, consisting of âˆ¼ 20,000 probes that target 1,465 loci across both families, has high locus recovery and phylogenetic utility. Phylogenetic analyses recovered the monophyly of major clades of Onychophora and revealed a novel lineage from the Neotropics that challenges our current understanding of onychophoran biogeographic endemicity. This new resource could drastically increase the power of molecular datasets and potentially allow access to genomic scale data from archival museum specimens to further tackle the issues exasperating onychophoran systematics.

A conserved polar residue plays a critical role in mismatch detection in A-family DNA polymerases.

In A-family DNA polymerases (dPols), a functional 3'-5' exonuclease activity is known to proofread newly synthesized DNA. The identification of a mismatch in substrate DNA leads to transfer of the primer strand from the polymerase active site to the exonuclease active site. To shed more light regarding the mechanism responsible for the detection of mismatches, we have utilized DNA polymerase 1 from Aquifex pyrophilus (ApPol1). The enzyme synthesized DNA with high fidelity and exhibited maximal exonuclease activity with DNA substrates bearing mismatches at the -2 and - 3 positions. The crystal structure of apo-ApPol1 was utilized to generate a computational model of the functional ternary complex of this enzyme. The analysis of the model showed that N332 forms interactions with minor groove atoms of the base pairs at the -2 and - 3 positions. The majority of known A-family dPols show the presence of Asn at a position equivalent to N332. The N332L mutation led to a decrease in the exonuclease activity for representative purine-pyrimidine, and pyrimidine-pyrimidine mismatches at -2 and - 3 positions, respectively. Overall, our findings suggest that conserved polar residues located towards the minor groove may facilitate the detection of position-specific mismatches to enhance the fidelity of DNA synthesis.

Nematode histone H2A variant evolution reveals diverse histories of retention and loss and evidence for conserved core-like variant histone genes.

Histone variants are paralogs that replace canonical histones in nucleosomes, often imparting novel functions. However, how histone variants arise and evolve is poorly understood. Reconstruction of histone protein evolution is challenging due to large differences in evolutionary rates across gene lineages and sites. Here we used intron position data from 108 nematode genomes in combination with amino acid sequence data to find disparate evolutionary histories of the three H2A variants found in Caenorhabditis elegans: the ancient H2A.ZHTZ-1, the sperm-specific HTAS-1, and HIS-35, which differs from the canonical S-phase H2A by a single glycine-to-alanine C-terminal change. Although the H2A.ZHTZ-1 protein sequence is highly conserved, its gene exhibits recurrent intron gain and loss. This pattern suggests that specific intron sequences or positions may not be important to H2A.Z functionality. For HTAS-1 and HIS-35, we find variant-specific intron positions that are conserved across species. Patterns of intron position conservation indicate that the sperm-specific variant HTAS-1 arose more recently in the ancestor of a subset of Caenorhabditis species, while HIS-35 arose in the ancestor of Caenorhabditis and its sister group, including the genus Diploscapter. HIS-35 exhibits gene retention in some descendent lineages but gene loss in others, suggesting that histone variant use or functionality can be highly flexible. Surprisingly, we find the single amino acid differentiating HIS-35 from core H2A is ancestral and common across canonical Caenorhabditis H2A sequences. Thus, we speculate that the role of HIS-35 lies not in encoding a functionally distinct protein, but instead in enabling H2A expression across the cell cycle or in distinct tissues. This work illustrates how genes encoding such partially-redundant functions may be advantageous yet relatively replaceable over evolutionary timescales, consistent with the patchwork pattern of retention and loss of both genes. Our study shows the utility of intron positions for reconstructing evolutionary histories of gene families, particularly those undergoing idiosyncratic sequence evolution.

Row1, a member of a new family of conserved fungal proteins involved in infection, is required for appressoria functionality in Ustilago maydis.

The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with nonmelanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria-specific protein of Ustilago maydis that localizes to membrane and secretory vesicles. Deletion of row1 decreases appressoria formation and plant penetration, thereby reducing virulence. Specifically, the Δrow1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the Δrow1 mutant has secretion defects. We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family. Our data suggest that Row1 could modify the chitin-glucan matrix of the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration.

Convergent accelerated evolution of mammal-specific conserved non-coding elements in hibernators.

Mammals maintain their body temperature, yet hibernators can temporarily lower their metabolic rate as an energy-saving strategy. It has been proposed that hibernators evolved independently from homeotherms, and it is possible that the convergent evolution of hibernation involved common genomic changes among hibernator-lineages. Since hibernation is a seasonal trait, the evolution of gene regulatory regions in response to changes in season may have been important for the acquisition of hibernation traits. High-frequency accumulation of mutations in conserved non-coding elements (CNEs) could, in principle, alter the expression of neighboring genes and thereby contribute to the acquisition of new traits. To address this possibility, we performed a comparative genomic analysis of mammals to identify accelerated CNEs commonly associated with hibernation. We found that accelerated CNEs are common to hibernator-lineages and could be involved with hibernation. We also found that common factors of genes that located near accelerated CNEs and are differentially expressed between normal and hibernation periods related to gene regulation and cell-fate determination. It suggests that the molecular mechanisms controlling hibernation have undergone convergent evolution. These results help broaden our understanding of the genetic adaptations that facilitated hibernation in mammals and may offer insights pertaining to stress responses and energy conservation.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation.

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5' end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A-H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies.

A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice.

Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.

Conserved genes regulating human sex differentiation, gametogenesis and fertilization.

The study of the functional genome in mice and humans has been instrumental for describing the conserved molecular mechanisms regulating human reproductive biology, and for defining the etiologies of monogenic fertility disorders. Infertility is a reproductive disorder that includes various conditions affecting a couple's ability to achieve a healthy pregnancy. Recent advances in next-generation sequencing and CRISPR/Cas-mediated genome editing technologies have facilitated the identification and characterization of genes and mechanisms that, if affected, lead to infertility. We report established genes that regulate conserved functions in fundamental reproductive processes (e.g., sex determination, gametogenesis, and fertilization). We only cover genes the deletion of which yields comparable fertility phenotypes in both rodents and humans. In the case of newly-discovered genes, we report the studies demonstrating shared cellular and fertility phenotypes resulting from loss-of-function mutations in both species. Finally, we introduce new model systems for the study of human reproductive biology and highlight the importance of studying human consanguineous populations to discover novel monogenic causes of infertility. The rapid and continuous screening and identification of putative genetic defects coupled with an efficient functional characterization in animal models can reveal novel mechanisms of gene function in human reproductive tissues.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.