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Discovery of target‐binding molecules, such as aptamers and peptides, is usually performed with the use of high‐throughput experimental screening methods. These methods typically generate large datasets of sequences of target‐binding molecules, which can be enriched with high affinity binders. However, the identification of the highest affinity binders from these large datasets often requires additional low‐throughput experiments or other approaches. Bioinformatics‐based analyses could be helpful to better understand these large datasets and identify the parts of the sequence space enriched with high affinity binders. BinderSpace is an open‐source Python package that performs motif analysis, sequence space visualization, clustering analyses, and sequence extraction from clusters of interest. The motif analysis, resulting in text‐based and visual output of motifs, can also provide heat maps of previously measured user‐defined functional properties for all the motif‐containing molecules. Users can also run principal component analysis (PCA) and t‐distributed stochastic neighbor embedding (t‐SNE) analyses on whole datasets and on motif‐related subsets of the data. Functionally important sequences can also be highlighted in the resulting PCA and t‐SNE maps. If points (sequences) in two‐dimensional maps in PCA or t‐SNE space form clusters, users can perform clustering analyses on their data, and extract sequences from clusters of interest. We demonstrate the use of BinderSpace on a dataset of oligonucleotides binding to single‐wall carbon nanotubes in the presence and absence of a bioanalyte, and on a dataset of cyclic peptidomimetics binding to bovine carbonic anhydrase protein. BinderSpace is openly accessible to the public via the GitHub website: https://github.com/vukoviclab/BinderSpace. [ABSTRACT FROM AUTHOR]

The 1-aminocyclopropane-1-carboxylic acid (ACC) pathway that synthesizes ethylene is shared in seed plants, fungi and probably other organisms. However, the evolutionary relationship of the key enzyme ACC oxidase (ACO) in the pathway among organisms remains unknown. Herein, we cloned, expressed and characterized five ACOs from the straw mushroom (Volvariella volvacea) and the oyster mushroom (Pleurotus ostreatus): VvACO1-4 and PoACO. The five mushroom ACOs and the previously identified AbACO of the button mushroom contained all three conserved residues that bound to Fe(II) in plant ACOs. They also had variable residues that were conserved and bound to ascorbate and bicarbonate in plant ACOs and harbored only 1–2 of the five conserved ACO motifs in plant ACOs. Particularly, VvACO2 and AbACO had only one ACO motif 2. Additionally, VvACO4 shared 44.23% sequence identity with the cyanobacterium Hapalosiphon putative functional ACO. Phylogenetic analysis showed that the functional ACOs of monocotyledonous and dicotyledonous plants co-occurred in Type I, Type II and Type III, while putative functional gymnosperm ACOs also appeared in Type III. The putative functional bacterial ACO, functional fungi and slime mold ACOs were clustered in ancestral Type IV. These results indicate that ACO motif 2, ACC and Fe(II) are essential for ACO activity. The ACOs of the other organisms may come from the horizontal transfer of fungal ACOs, which were found ordinarily in basidiomycetes. It is mostly the first case for the horizontal gene transfers from fungi to seed plants. The horizontal transfer of ACOs from fungi to plants probably facilitates the fungal-plant symbioses, plant–land colonization and further evolution to form seeds. [ABSTRACT FROM AUTHOR]

The 1-aminocyclopropane-1-carboxylic acid (ACC) pathway that synthesizes ethylene is shared in seed plants, fungi and probably other organisms. However, the evolutionary relationship of the key enzyme ACC oxidase (ACO) in the pathway among organisms remains unknown. Herein, we cloned, expressed and characterized five ACOs from the straw mushroom (Volvariella volvacea) and the oyster mushroom (Pleurotus ostreatus): VvACO1-4 and PoACO. The five mushroom ACOs and the previously identified AbACO of the button mushroom contained all three conserved residues that bound to Fe(II) in plant ACOs. They also had variable residues that were conserved and bound to ascorbate and bicarbonate in plant ACOs and harbored only 1–2 of the five conserved ACO motifs in plant ACOs. Particularly, VvACO2 and AbACO had only one ACO motif 2. Additionally, VvACO4 shared 44.23% sequence identity with the cyanobacterium Hapalosiphon putative functional ACO. Phylogenetic analysis showed that the functional ACOs of monocotyledonous and dicotyledonous plants co-occurred in Type I, Type II and Type III, while putative functional gymnosperm ACOs also appeared in Type III. The putative functional bacterial ACO, functional fungi and slime mold ACOs were clustered in ancestral Type IV. These results indicate that ACO motif 2, ACC and Fe(II) are essential for ACO activity. The ACOs of the other organisms may come from the horizontal transfer of fungal ACOs, which were found ordinarily in basidiomycetes. It is mostly the first case for the horizontal gene transfers from fungi to seed plants. The horizontal transfer of ACOs from fungi to plants probably facilitates the fungal-plant symbioses, plant–land colonization and further evolution to form seeds.

There is increasing interest in developing diagnostics that discriminate individual mutagenic mechanisms in a range of applications that include identifying population-specific mutagenesis and resolving distinct mutation signatures in cancer samples. Analyses for these applications assume that mutagenic mechanisms have a distinct relationship with neighboring bases that allows them to be distinguished. Direct support for this assumption is limited to a small number of simple cases, e.g., CpG hypermutability. We have evaluated whether the mechanistic origin of a point mutation can be resolved using only sequence context for a more complicated case. We contrasted single nucleotide variants originating from the multitude of mutagenic processes that normally operate in the mouse germline with those induced by the potent mutagen N-ethyl-N-nitrosourea (ENU). The considerable overlap in the mutation spectra of these two samples make this a challenging problem. Employing a new, robust log-linear modeling method, we demonstrate that neighboring bases contain information regarding point mutation direction that differs between the ENU-induced and spontaneous mutation variant classes. A logistic regression classifier exhibited strong performance at discriminating between the different mutation classes. Concordance between the feature set of the best classifier and information content analyses suggest our results can be generalized to other mutation classification problems. We conclude that machine learning can be used to build a practical classification tool to identify the mutation mechanism for individual genetic variants. Software implementing our approach is freely available under an open-source license. [ABSTRACT FROM AUTHOR]

Superoxide dismutases (SODs), as a family of metalloenzymes related to the removal of reactive oxygen species (ROS), have not previously been investigated at genome-wide level in tea plant. In this study, 10 CsSOD genes were identified in tea plant genome, including 7 Cu/Zn-SODs (CSDs), 2 Fe-SODs (FSDs) and one Mn-SOD (MSD), and phylogenetically classified in three subgroups, respectively. Physico-chemical characteristic, conserved motifs and potential protein interaction analyses about CsSOD proteins were carried out. Exon-intron structures and codon usage bias about CsSOD genes were also examined. Exon-intron structures analysis revealed that different CsSOD genes contained various number of introns. On the basis of the prediction of regulatory miRNAs of CsSODs, a modification 5' RNA ligase-mediated (RLM)-RACE was performed and validated that csn-miR398a-3p-1 directly cleaves CsCSD4. By prediction of cis-acting elements, the expression patterns of 10 CsSOD genes and their regulatory miRNAs were detected under cold, drought, exogenous methyl jasmonate (MeJA) and gibberellin (GA3) treatments. The results showed that most of CsSODs except for CsFSD2 were induced under cold stress and CsCSDs may play primary roles under drought stress; exogenous GA3 and MeJA could also stimulated/inhibited distinct CsSODs at different stages. In addition, we found that csn-miR398a-3p-1 negatively regulated the expression of CsCSD4 may be a crucial regulatory mechanism under cold stress. This study provides a certain basis for the studies about stress resistance in tea plants, even provide insight into comprehending the classification, evolution, diverse functions and influencing factors of expression patterns for CsSOD genes. [ABSTRACT FROM AUTHOR]

The formation of the sea urchin spicule skeleton requires the participation of hydrogel-forming protein families that regulate mineral nucleation and nanoparticle assembly processes that give rise to the spicule. However, the structure and molecular behavior of these proteins is not well established, and thus our ability to understand this process is hampered. We embarked on a study of sea urchin spicule proteins using a combination of biophysical and bioinformatics techniques. Our biophysical findings indicate that recombinant variants of the two most studied spicule matrix proteins, SpSM50 and SpSM30B/C (S. purpuratus) have a conformational landscape that include a C-terminal random coil/intrinsically disordered MAPQG sequence coupled to a conserved, folded N-terminal C-type lectin-like (CTLL) domain, with SpSM50 > SpSM30B/C with regard to intrinsic disorder. Both proteins possess solvent-accessible unfolded MAQPG sequence regions where Asn, Gln, and Arg residues may be accessible for protein hydrogel interactions with water molecules. Our bioinformatics study included seven other spicule matrix proteins where we note similarities between these proteins and rare, unusual proteins that possess folded and unfolded traits. Moreover, spicule matrix proteins possess three types of sequences: intrinsically disordered, amyloid-like, and folded protein-protein interactive. Collectively these reactive domains would be capable of driving protein assembly and hydrogel formation. Interestingly, three types of global conformations are predicted for the nine member protein set, wherein we note variations in the arrangement of intrinsically disordered and interactive globular domains. These variations may reflect species-specific requirements for spiculogenesis. We conclude that the molecular landscape of spicule matrix protein families enables them to function as hydrogelators, nucleators, and assemblers of mineral nanoparticles. [ABSTRACT FROM AUTHOR]

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most ‘ancient’ ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex. [ABSTRACT FROM AUTHOR]

Telomeres are nucleo-protein structures that protect the ends of eukaryotic chromosomes. They are not completely synthesized during DNA replication and are elongated by specific mechanisms. The structure of the telomeres and the elongation mechanism have not been determined in Dictyostelium discoideum. This organism presents extrachromosomal palindromic elements containing two copies of the rDNA, also present at the end of the chromosomes. In this article the structure of the terminal region of the rDNA is shown to consist of repetitions of the A(G)n sequence where the number of Gs is variable. These repeats extend as a 3’ single stranded region. The G-rich region is preceded by four tandem repetitions of two different DNA motifs. D. discoideum telomere reverse transcriptase homologous protein (TERTHP) presented RNase-sensitive enzymatic activity and was required to maintain telomere structure since terthp-mutant strains presented reorganizations of the DNA terminal regions. These modifications were different in several terthp-mutants and changed with their prolonged culture and subcloning. However, the terthp gene is not essential for D. discoideum proliferation. Telomeres could be maintained in terthp-mutant strains by homologous recombination mechanisms such as ALT (Alternative Lengthening of Telomeres) or HAATI (heterochromatin amplification-mediated and telomerase-independent). In agreement with this hypothesis, the expression of mRNAs coding for several proteins involved in homologous recombination was induced in terthp-mutant strains. Extrachromosomal rDNA could serve as substrate in these DNA homologous recombination reactions. [ABSTRACT FROM AUTHOR]

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5′ end of env. [ABSTRACT FROM AUTHOR]

Neuropeptides are a class of bioactive peptides shown to be involved in various physiological processes, including metabolism, development, and reproduction. Although neuropeptide candidates have been predicted from genomic and transcriptomic data, comprehensive characterization of neuropeptide repertoires remains a challenge owing to their small size and variable sequences. De novo prediction of neuropeptides from genome or transcriptome data is difficult and usually only efficient for those peptides that have identified orthologs in other animal species. Recent peptidomics technology has enabled systematic structural identification of neuropeptides by using the combination of liquid chromatography and tandem mass spectrometry. However, reliable identification of naturally occurring peptides using a conventional tandem mass spectrometry approach, scanning spectra against a protein database, remains difficult because a large search space must be scanned due to the absence of a cleavage enzyme specification. We developed a pipeline consisting of in silico prediction of candidate neuropeptides followed by peptide-spectrum matching. This approach enables highly sensitive and reliable neuropeptide identification, as the search space for peptide-spectrum matching is highly reduced. Nematostella vectensis is a basal eumetazoan with one of the most ancient nervous systems. We scanned the Nematostella protein database for sequences displaying structural hallmarks typical of eumetazoan neuropeptide precursors, including amino- and carboxyterminal motifs and associated modifications. Peptide-spectrum matching was performed against a dataset of peptides that are cleaved in silico from these putative peptide precursors. The dozens of newly identified neuropeptides display structural similarities to bilaterian neuropeptides including tachykinin, myoinhibitory peptide, and neuromedin-U/pyrokinin, suggesting these neuropeptides occurred in the eumetazoan ancestor of all animal species. [ABSTRACT FROM AUTHOR]

The deep-sea chemosynthetic environment is one of the most extreme environments on the Earth, with low oxygen, high hydrostatic pressure and high levels of toxic substances. Species of the family Vesicomyidae are among the dominant chemosymbiotic bivalves found in this harsh habitat. Mitochondria play a vital role in oxygen usage and energy metabolism; thus, they may be under selection during the adaptive evolution of deep-sea vesicomyids. In this study, the mitochondrial genome (mitogenome) of the vesicomyid bivalve Calyptogena marissinica was sequenced with Illumina sequencing. The mitogenome of C. marissinica is 17,374 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes (rrnS and rrnL) and 22 transfer RNA genes. All of these genes are encoded on the heavy strand. Some special elements, such as tandem repeat sequences, “G(A)nT” motifs and AT-rich sequences, were observed in the control region of the C. marissinica mitogenome, which is involved in the regulation of replication and transcription of the mitogenome and may be helpful in adjusting the mitochondrial energy metabolism of organisms to adapt to the deep-sea chemosynthetic environment. The gene arrangement of protein-coding genes was identical to that of other sequenced vesicomyids. Phylogenetic analyses clustered C. marissinica with previously reported vesicomyid bivalves with high support values. Positive selection analysis revealed evidence of adaptive change in the mitogenome of Vesicomyidae. Ten potentially important adaptive residues were identified, which were located in cox1, cox3, cob, nad2, nad4 and nad5. Overall, this study sheds light on the mitogenomic adaptation of vesicomyid bivalves that inhabit the deep-sea chemosynthetic environment. [ABSTRACT FROM AUTHOR]

Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances. [ABSTRACT FROM AUTHOR]

Members of the plant Heme Activator Protein (HAP) or NUCLEAR FACTOR Y (NF-Y) are trimeric transcription factor complexes composed of the NF-YA, NF-YB and NF-YC subfamilies. They bind to the CCAAT box in the promoter regions of the target genes and regulate gene expressions. Plant NF-Ys were reported to be involved in adaptation to several abiotic stresses as well as in development. In silico analysis of Sorghum bicolor genome resulted in the identification of a total of 42 NF-Y genes, among which 8 code for the SbNF-YA, 19 for SbNF-YB and 15 for the SbNF-YC subunits. Analysis was also performed to characterize gene structures, chromosomal distribution, duplication status, protein subcellular localizations, conserved motifs, ancestral protein sequences, miRNAs and phylogenetic tree construction. Phylogenetic relationships and ortholog predictions displayed that sorghum has additional NF-YB genes with unknown functions in comparison with Arabidopsis. Analysis of promoters revealed that they harbour many stress-related cis-elements like ABRE and HSE, but surprisingly, DRE and MYB elements were not detected in any of the subfamilies. SbNF-YA1, 2, and 6 were found upregulated under 200 mM salt and 200 mM mannitol stresses. While NF-YA7 appeared associated with high temperature (40°C) stress, NF-YA8 was triggered by both cold (4°C) and high temperature stresses. Among NF-YB genes, 7, 12, 15, and 16 were induced under multiple stress conditions such as salt, mannitol, ABA, cold and high temperatures. Likewise, NF-YC 6, 11, 12, 14, and 15 were enhanced significantly in a tissue specific manner under multiple abiotic stress conditions. Majority of the mannitol (drought)-inducible genes were also induced by salt, high temperature stresses and ABA. Few of the high temperature stress-induced genes are also induced by cold stress (NF-YA2, 4, 6, 8, NF-YB2, 7, 10, 11, 12, 14, 16, 17, NF-YC4, 6, 12, and 13) thus suggesting a cross talk among them. This work paves the way for investigating the roles of diverse sorghum NF-Y proteins during abiotic stress responses and provides an insight into the evolution of diverse NF-Y members. [ABSTRACT FROM AUTHOR]

At some point early in the vertebrate lineage, two whole genome duplication events (1R, 2R) took place that allowed for the diversification and sub-/neo-functionalization of the glycoprotein hormones (GpHs). All jawed vertebrates possess the GpHs luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH), each of which are heterodimers with a common alpha subunit and unique beta subunits. In 2002, a novel glycoprotein hormone named thyrostimulin was described to have unique GpA2 and GpB5 subunits that were homologous to the vertebrate alpha and beta subunits. The presence of GpA2 and GpB5 in representative protostomes and deuterostomes indicates their ancestry in the GpH family. There are several reports of GpH subunit evolution, but none have included GpA2 and GpB5 for species in each major vertebrate class. Thus, we addressed the ancestry of two paralogous GpB5 subunits (GpB5a and GpB5b) that were previously only recognized in two teleost species. Our search for orthologous GpB5a and GpB5b sequences in representative vertebrates and phylogenetic analysis, in addition to the currently published evolutionary scenarios of the GpH family, supports that GpB5a and GpB5b are paralogs that arose from the first vertebrate whole genome duplication event (1R). Syntenic analysis supports lineage specific losses of GpB5a in chondrichthyes, basal actinopterygians, and tetrapods, and retention in coelacanth and teleosts. Additionally, we were unable to identify GpA2 transcripts from tilapia mRNA, suggesting that this species does not produce heterodimeric thyrostimulin. While the conserved or even species-specific functional role of thyrostimulin or its individual subunits are still unknown in vertebrates, the analyses presented here provide context for future studies on the functional divergence of the GpH family. [ABSTRACT FROM AUTHOR]

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants. [ABSTRACT FROM AUTHOR]

Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism’s complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors. [ABSTRACT FROM AUTHOR]

Allatoregulatory neuropeptides are multifunctional proteins that take part in the synthesis and secretion of juvenile hormones. In insects, allatostatins are inhibitors of juvenile hormone biosynthesis in the corpora allata while allatotropins, act as stimulators. By quantitative real-time PCR, we analyzed the gene expression of allatostatin A (PtASTA), allatostatin B (PtASTB), allatostatin C (PtASTC), allatotropin (PtAT) and their receptors (PtASTA-R, PtASTB-R, PtASTC-R, PtAT-R) in various tissues in different age groups of female spiders. In the presented manuscript, the presence of allatostatin A, allatostatin C, and allatotropin are reported in females of the spider P. tepidariorum. The obtained results indicated substantial differences in gene expression levels for allatoregulatory neuropeptides and their receptors in the different tissues. Additionally, the gene expression levels also varied depending on the female age. Strong expression was observed coinciding with sexual maturation in the neuroendocrine and nervous system, and to a lower extent in the digestive tissues and ovaries. Reverse trends were observed for the expression of genes encoding the receptors of these neuropeptides. In conclusion, our study is the first hint that the site of synthesis and secretion is fulfilled by similar structures as observed in other arthropods. In addition, the results of the analysis of spider physiology give evidence that the general functions like regulation of the juvenile hormone synthesis, regulation of the digestive tract and ovaries action, control of vitellogenesis process by the neuropeptides seem to be conserved among arthropods and are the milestone to future functional studies. [ABSTRACT FROM AUTHOR]

Despite the importance of microbial activity in mobilizing arsenic in groundwater aquifers, the phylogenetic distribution of contributing microbial metabolisms is understudied. Groundwater samples from Ohio aquifers were analyzed using metagenomic sequencing to identify functional potential that could drive arsenic cycling, and revealed mechanisms for direct (i.e., Ars system) and indirect (i.e., iron reduction) arsenic mobilization in all samples, despite differing geochemical conditions. Analyses of 194 metagenome-assembled genomes (MAGs) revealed widespread functionality related to arsenic mobilization throughout the bacterial tree of life. While arsB and arsC genes (components of an arsenic resistance system) were found in diverse lineages with no apparent phylogenetic bias, putative aioA genes (aerobic arsenite oxidase) were predominantly identified in Methylocystaceae MAGs. Both previously described and undescribed respiratory arsenate reduction potential via arrA was detected in Betaproteobacteria, Deltaproteobacteria, and Nitrospirae MAGs, whereas sulfate reduction potential was primarily limited to members of the Deltaproteobacteria and Nitrospirae. Lastly, iron reduction potential was detected in the Ignavibacteria, Deltaproteobacteria, and Nitrospirae. These results expand the phylogenetic distribution of taxa that may play roles in arsenic mobilization in subsurface systems. Specifically, the Nitrospirae are a much more functionally diverse group than previously assumed and may play key biogeochemical roles in arsenic-contaminated ecosystems. [ABSTRACT FROM AUTHOR]

Antibodies are essential to functional immunity, yet the epitopes targeted by antibody repertoires remain largely uncharacterized. To aid in characterization, we developed a generalizable strategy to predict antibody-binding epitopes within individual proteins and entire proteomes. Specifically, we selected antibody-binding peptides for 273 distinct sera out of a random library and identified the peptides using next-generation sequencing. To predict antibody-binding epitopes and the antigens from which these epitopes were derived, we tiled the sequences of candidate antigens into short overlapping subsequences of length k (k-mers). We used the enrichment over background of these k-mers in the antibody-binding peptide dataset to predict antibody-binding epitopes. As a positive control, we used this approach, termed K-mer Tiling of Protein Epitopes (K-TOPE), to predict epitopes targeted by monoclonal and polyclonal antibodies of well-characterized specificity, accurately recovering their known epitopes. K-TOPE characterized a commonly targeted antigen from Rhinovirus A, predicting four epitopes recognized by antibodies present in 87% of sera (n = 250). An analysis of 2,908 proteins from 400 viral taxa that infect humans predicted seven enterovirus epitopes and five Epstein-Barr virus epitopes recognized by >30% of specimens. Analysis of Staphylococcus and Streptococcus proteomes similarly predicted 22 epitopes recognized by >30% of specimens. Twelve of these common viral and bacterial epitopes agreed with previously mapped epitopes with p-values < 0.05. Additionally, we predicted 30 HSV2-specific epitopes that were 100% specific against HSV1 in novel and previously reported antigens. Experimentally validating these candidate epitopes could help identify diagnostic biomarkers, vaccine components, and therapeutic targets. The K-TOPE approach thus provides a powerful new tool to elucidate the organisms, antigens, and epitopes targeted by human antibody repertoires. [ABSTRACT FROM AUTHOR]

The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite. [ABSTRACT FROM AUTHOR]

Predicting RNA-binding protein (RBP) specificity is important for understanding gene expression regulation and RNA-mediated enzymatic processes. It is widely believed that RBP binding specificity is determined by both the sequence and structural contexts of RNAs. Existing approaches, including traditional machine learning algorithms and more recently, deep learning models, have been extensively applied to integrate RNA sequence and its predicted or experimental RNA structural probabilities for improving the accuracy of RBP binding prediction. Such models were trained mostly on the large-scale in vitro datasets, such as the RNAcompete dataset. However, in RNAcompete, most synthetic RNAs are unstructured, which makes machine learning methods not effectively extract RBP-binding structural preferences. Furthermore, RNA structure may be variable or multi-modal according to both theoretical and experimental evidence. In this work, we propose ThermoNet, a thermodynamic prediction model by integrating a new sequence-embedding convolutional neural network model over a thermodynamic ensemble of RNA secondary structures. First, the sequence-embedding convolutional neural network generalizes the existing k-mer based methods by jointly learning convolutional filters and k-mer embeddings to represent RNA sequence contexts. Second, the thermodynamic average of deep-learning predictions is able to explore structural variability and improves the prediction, especially for the structured RNAs. Extensive experiments demonstrate that our method significantly outperforms existing approaches, including RCK, DeepBind and several other recent state-of-the-art methods for predictions on both in vitro and in vivo data. The implementation of ThermoNet is available at . [ABSTRACT FROM AUTHOR]

The availability of large amounts of high-throughput genomic, transcriptomic and epigenomic data has provided opportunity to understand regulation of the cellular transcriptome with an unprecedented level of detail. As a result, research has advanced from identifying gene expression patterns associated with particular conditions to elucidating signalling pathways that regulate expression. There are over 1,000 transcription factors (TFs) in vertebrates that play a role in this regulation. Determining which of these are likely to be controlling a set of genes can be assisted by computational prediction, utilising experimentally verified binding site motifs. Here we present CiiiDER, an integrated computational toolkit for transcription factor binding analysis, written in the Java programming language, to make it independent of computer operating system. It is operated through an intuitive graphical user interface with interactive, high-quality visual outputs, making it accessible to all researchers. CiiiDER predicts transcription factor binding sites (TFBSs) across regulatory regions of interest, such as promoters and enhancers derived from any species. It can perform an enrichment analysis to identify TFs that are significantly over- or under-represented in comparison to a bespoke background set and thereby elucidate pathways regulating sets of genes of pathophysiological importance. [ABSTRACT FROM AUTHOR]

There has been much effort to prioritize genomic variants with respect to their impact on “function”. However, function is often not precisely defined: sometimes it is the disease association of a variant; on other occasions, it reflects a molecular effect on transcription or epigenetics. Here, we coupled multiple genomic predictors to build GRAM, a GeneRAlized Model, to predict a well-defined experimental target: the expression-modulating effect of a non-coding variant on its associated gene, in a transferable, cell-specific manner. Firstly, we performed feature engineering: using LASSO, a regularized linear model, we found transcription factor (TF) binding most predictive, especially for TFs that are hubs in the regulatory network; in contrast, evolutionary conservation, a popular feature in many other variant-impact predictors, has almost no contribution. Moreover, TF binding inferred from in vitro SELEX is as effective as that from in vivo ChIP-Seq. Second, we implemented GRAM integrating only SELEX features and expression profiles; thus, the program combines a universal regulatory score with an easily obtainable modifier reflecting the particular cell type. We benchmarked GRAM on large-scale MPRA datasets, achieving AUROC scores of 0.72 in GM12878 and 0.66 in a multi-cell line dataset. We then evaluated the performance of GRAM on targeted regions using luciferase assays in the MCF7 and K562 cell lines. We noted that changing the insertion position of the construct relative to the reporter gene gave very different results, highlighting the importance of carefully defining the exact prediction target of the model. Finally, we illustrated the utility of GRAM in fine-mapping causal variants and developed a practical software pipeline to carry this out. In particular, we demonstrated in specific examples how the pipeline could pinpoint variants that directly modulate gene expression within a larger linkage-disequilibrium block associated with a phenotype of interest (e.g., for an eQTL). [ABSTRACT FROM AUTHOR]

It is extremely important to identify transcription factor binding sites (TFBSs). Some TFBSs are proposed to be bipartite motifs known as two-block motifs separated by gap sequences with variable lengths. While position weight matrix (PWM) is commonly used for the representation and prediction of TFBSs, dinucleotide weight matrix (DWM) enables expression of the interdependencies of neighboring bases. By incorporating DWM into the detection of bipartite motifs, we have developed a novel tool for ab initio motif detection, DIpartite (bipartite motif detection tool based on dinucleotide weight matrix) using a Gibbs sampling strategy and the minimization of Shannon’s entropy. DIpartite predicts the bipartite motifs by considering the interdependencies of neighboring positions, that is, DWM. We compared DIpartite with other available alternatives by using test datasets, namely, of CRP in E. coli, sigma factors in B. subtilis, and promoter sequences in humans. We have developed DIpartite for the detection of TFBSs, particularly bipartite motifs. DIpartite enables ab initio prediction of conserved motifs based on not only PWM, but also DWM. We evaluated the performance of DIpartite by comparing it with freely available tools, such as MEME, BioProspector, BiPad, and AMD. Taken the obtained findings together, DIpartite performs equivalently to or better than these other tools, especially for detecting bipartite motifs with variable gaps. DIpartite requires users to specify the motif lengths, gap length, and PWM or DWM. DIpartite is available for use at . [ABSTRACT FROM AUTHOR]

Intercellular communication in adjacent cell layers determines cell fate and polarity, thus orchestrating tissue specification and differentiation. Here we use the maize stomatal apparatus as a model to investigate cell fate determination. Mutations in ZmBZU2 (ii2, bzu2) confer a complete absence of subsidiary cells (SCs) and normal guard cells (GCs), leading to failure of formation of mature stomatal complexes. Nuclear polarization and actin accumulation at the interface between subsidiary mother cells (SMCs) and guard mother cells (GMCs), an essential pre-requisite for asymmetric cell division, did not occur in Zmbzu2 mutants. ZmBZU2 encodes a basic helix-loop-helix (bHLH) transcription factor, which is an ortholog of AtMUTE in Arabidopsis (BZU2/ZmMUTE). We found that a number of genes implicated in stomatal development are transcriptionally regulated by BZU2/ZmMUTE. In particular, BZU2/ZmMUTE directly binds to the promoters of PAN1 and PAN2, two early regulators of protodermal cell fate and SMC polarization, consistent with the low levels of transcription of these genes observed in bzu2-1 mutants. BZU2/ZmMUTE has the cell-to-cell mobility characteristic similar to that of BdMUTE in Brachypodium distachyon. Unexpectedly, BZU2/ZmMUTE is expressed in GMC from the asymmetric division stage to the GMC division stage, and especially in the SMC establishment stage. Taken together, these data imply that BZU2/ZmMUTE is required for early events in SMC polarization and differentiation as well as for the last symmetrical division of GMCs to produce the two GCs, and is a master determinant of the cell fate of its neighbors through cell-to-cell communication. [ABSTRACT FROM AUTHOR]

Membrane-protein design is an exciting and increasingly successful research area which has led to landmarks including the design of stable and accurate membrane-integral proteins based on coiled-coil motifs. Design of topologically more complex proteins, such as most receptors, channels, and transporters, however, demands an energy function that balances contributions from intra-protein contacts and protein-membrane interactions. Recent advances in water-soluble all-atom energy functions have increased the accuracy in structure-prediction benchmarks. The plasma membrane, however, imposes different physical constraints on protein solvation. To understand these constraints, we recently developed a high-throughput experimental screen, called dsTβL, and inferred apparent insertion energies for each amino acid at dozens of positions across the bacterial plasma membrane. Here, we express these profiles as lipophilicity energy terms in Rosetta and demonstrate that the new energy function outperforms previous ones in modelling and design benchmarks. Rosetta ab initio simulations starting from an extended chain recapitulate two-thirds of the experimentally determined structures of membrane-spanning homo-oligomers with <2.5Å root-mean-square deviation within the top-predicted five models (available online: ). Furthermore, in two sequence-design benchmarks, the energy function improves discrimination of stabilizing point mutations and recapitulates natural membrane-protein sequences of known structure, thereby recommending this new energy function for membrane-protein modelling and design. [ABSTRACT FROM AUTHOR]

Sigma factors are key proteins that mediate the recruitment of RNA polymerase to the promoter regions of genes, for the initiation of bacterial transcription. Multiple sigma factors in a bacterium selectively recognize their cognate promoter sequences, thereby inducing the expression of their own regulons. In this paper, we report the crystal structure of the σ4 domain of Bacillus subtilis SigW bound to the -35 promoter element. Purine-specific hydrogen bonds of the -35 promoter element with the recognition helix α9 of the σ4 domain occurs at three nucleotides of the consensus sequence (G-35, A-34, and G’-31 in G-35A-34A-33A-32C-31C-30T-29). The hydrogen bonds of the backbone with the α7 and α8 of the σ4 domain occurs at G’-30. These results elucidate the structural basis of the selective recognition of the promoter by SigW. In addition, comparison of SigW structures complexed with the -35 promoter element or with anti-sigma RsiW reveals that DNA recognition and anti-sigma factor binding of SigW are mutually exclusive. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) infection is fatal in domesticated pigs, with a mortality rate approaching 100%. This may result in economic losses and threats to food security. Currently, there are no approved vaccines or antiviral therapies for ASFV. Therefore, in this study, we evaluated congocidine congeners and a tris-benzimidazole as potential inhibitors of ASFV transcription using an in silico approach. We applied redocking of congocidine and docking of its congeners and a tris-benzimidazole to a receptor containing B-DNA with AT-motifs as a target to mimic conserved ASFV late gene promoters. Subsequently, the binding scores of DNA-ligand docked complexes were evaluated and their binding affinity was estimated. Molecular dynamics (MD) simulation was then used to assess ligand behavior within the minor groove. From our results, it is evident the less toxic congocidine congeners and tris-benzimidazole could dock to AT-rich regions significantly. Additionally, the predicted binding affinities had suitable values comparable to other experimentally determined minor groove binders, MD simulation of the docked DNA-ligand complexes and subsequent molecular trajectory visualization further showed that the ligands remained embedded in the minor groove during the time course of simulation, indicating that these ligands may have potential applications in abrogating ASFV transcription. [ABSTRACT FROM AUTHOR]

Plant NLRs are modular immune receptors that trigger rapid cell death in response to attempted infection by pathogens. A highly conserved ucleotide-inding domain shared with PAF-1, various -proteins and ED-4 (NB-ARC domain) is proposed to act as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies of plant NLR NB-ARC domains have revealed functional similarities to mammalian homologues, and provided insight into potential mechanisms of regulation. However, further advances have been limited by difficulties in obtaining sufficient yields of protein suitable for structural and biochemical techniques. From protein expression screens in Escherichia coli and Sf9 insect cells, we defined suitable conditions to produce the NB-ARC domain from the tomato NLR NRC1. Biophysical analyses of this domain showed it is a folded, soluble protein. Structural studies revealed the NRC1 NB-ARC domain had co-purified with ADP, and confirmed predicted structural similarities between plant NLR NB-ARC domains and their mammalian homologues. [ABSTRACT FROM AUTHOR]

The lentiviral Rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 (HIV-1). The main function of Rev is to mediate the nuclear exportation of viral RNAs. To fulfill its function, Rev shuttles between the cytoplasm and the nucleus. The Jembrana disease virus (JDV), a lentivirus, is the etiologic agent of the Jembrana disease which was first described in Bali cattle in Indonesia in 1964. Despite the high mortality rate associated with JDV, this virus remains poorly studied. Herein the subcellular distribution of JDV Rev, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the protein were examined. JDV Rev fused to the enhanced green fluorescent protein (EGFP) predominantly localized to the cytoplasm and nucleolus of transfected cells, as determined by fluorescence microscopy analyses. Through transfection of a series of deletion mutants of JDV Rev, it was possible to localize the NLS/NoLS region between amino acids (aa) 74 to 105. By substituting basic residues with alanine within this sequence, we demonstrated that the JDV Rev NLS encompasses aa 76 to 86, and is exclusively composed of arginine residues, whereas a bipartite NoLS was observed for the first time in any retroviral Rev/Rev-like proteins. Finally, a NES was identified downstream of the NLS/NoLS and encompasses aa 116 to 128 of the JDV Rev protein. The JDV Rev NES was found to be of the protein kinase A inhibitor (PKI) class instead of the HIV-1 Rev class. It also corresponds to the most optimal consensus sequence of PKI NES and, as such, is novel among lentiviral Rev NES. [ABSTRACT FROM AUTHOR]

Angelica dahurica is a widely grown plant species with multiple uses, especially in the medical field. However, the frequent introduction of A. dahurica to new areas has made it difficult to distinguish between varieties. Simple sequence repeats (SSRs) detected based on transcriptome analyses are very useful for constructing genetic maps and analyzing genetic diversity. They are also relevant for the molecular marker-assisted breeding of A. dahurica. We identified 33,724 genic SSR loci based on transcriptome sequencing data. A total of 114 primer pairs were designed for the SSR loci and were tested for their specificity and diversity. Ten SSR loci in untranslated regions were ultimately selected. Subsequently, 56 A. dahurica ecotypes collected from different regions were analyzed. The SSR loci comprised 2–8 alleles, with a mean of 5.2 alleles per locus. The polymorphic information content value and Shannon’s information index were 0.6274–0.2702 (average of 0.4091) and 1.3040–0.5618 (average of 0.8475), respectively. Thus, the 10 novel SSRs identified in this study were almost in accordance with Harvey-Weinberg equilibrium and will be useful for analyzing A. dahurica genetic relationships. The results of this study confirm the potential value of transcriptome databases for the development of new SSR markers. [ABSTRACT FROM AUTHOR]

JMJD6 is a member of the Jumonji C domain containing enzymes that demethylate and/or hydroxylate substrate proteins. It is a multi-functional protein that has been implicated in disparate aspects of transcriptional and post-transcriptional control of gene expression, including but not limited to enhancer and promoter binding, release of paused RNA polymerase II, control of splicing, and interaction with the translation machinery. JMJD6 contributes to multiple aspects of animal development, including adipogenesis modeled in culture. We mutated proposed or characterized domains in the JMJD6 protein to better understand the requirement for JMJD6 in adipogenic differentiation. Mutation of JMJD6 amino acids that mediate binding of iron and 2-oxogluterate, which are required cofactors for enzymatic activity, had no impact on JMJD6 function, showing that catalytic activity is not required for JMJD6 contributions to adipogenic differentiation. In addition, we documented the formation of JMJD6 oligomers and showed that catalytic activity is not required for oligomerization, as has been reported previously. We also observed no effect of mutations in the sumoylation site and in the poly-serine stretch. In contrast, mutation of the AT hook-like structure, which mediates interaction with DNA and/or RNA, compromised JMJD6 function by blocking its ability to interact with chromatin at genes that express regulators of adipogenesis. The ability of JMJD6 to interact with nucleic acids may be a critical requirement for its function in adipogenic differentiation. The requirement for the AT hook-like domain and the lack of requirement for catalytic activity giving rise to the idea that co-activation of transcription by JMJD6 may be functioning as a scaffold protein that supports the interactions of other critical regulators. [ABSTRACT FROM AUTHOR]

Sulfotransferases (SOTs; EC 2.8.2.-), which are widespread from prokaryotes to eukaryotes, constitute a multi-protein family that plays crucial roles in plant growth, development and stress adaptation. However, this family has not been systemically investigated in Brassica rapa. Here, a genome-wide systemic analysis of SOT genes in B. rapa subsp. pekinensis, a globally cultivated vegetable, were conducted. We identified 56 SOT genes from the whole B. rapa genome using Arabidopsis SOT sequences as queries and classified them into nine groups, rather than the eight groups of previous research. 56 B. rapa SOT genes (BraSOTs) were distributed on all 10 chromosomes except for chromosome 5. Of these, 27 BraSOTs were distributed in seven clusters on five chromosomes (ChrA01, ChrA02, Chr03, ChrA07, and Chr09). Among the BraSOT proteins, 48 had only one SOT_1 domain and 6 had two, while 2 had one SOT_3 domain. Additionally, 47 BraSOT proteins contained only known SOT domains. The remaining nine proteins, five in group-VIII and two in group-IX, contained additional transmembrane domains. Specific motif regions I and IV for 3′-phosphoadenosine 5′-phosphosulfate binding were found in 41 BraSOT proteins. Introns were present in only 18 BraSOT genes, and all seven BraSOT genes in groups VIII and IX had more than three introns. To identify crucial SOTs mediating the response to abiotic stress in B. rapa, expression changes in 56 BraSOT genes were determined by quantitative RT-PCR after drought, salinity, and ABA treatments, and some BraSOT genes were associated with NaCl, drought and ABA stress, e.g. Bra017370, Bra009300, Bra027880. [ABSTRACT FROM AUTHOR]

Structural diversity in α-helical membrane proteins (MP) arises from variations in helix-helix crossings and contacts that may bias amino acid usage. Here, we reveal systematic changes in transmembrane amino acid frequencies (f) as a function of the number of helices (n). For eukarya, breaks in f(n) trends of packing (Ala, Gly and Pro), polar, and hydrophobic residues identify different MP assembly principles for 2≤n≤7, 8≤n≤12 and n≥13. In bacteria, the first f break already occurs after n = 6 in correlation to an earlier n peak in MP size distribution and dominance of packing over polar interactions. In contrast to the later n brackets, the integration levels of helix bundles continuously increased in the first, most populous brackets indicating the formation of single structural units (domains). The larger first bracket of eukarya relates to a balance of polar and packing interactions that enlarges helix-helix combinatorial possibilities (MP diversity). Between the evolutionary old, packing and new, polar residues f anti-correlations extend over all biological taxa, broadly ordering them according to evolutionary history and allowing f estimates for the earliest forms of life. Next to evolutionary history, the amino acid composition of MP is determined by size (n), proteome diversity, and effective amino acid cost. [ABSTRACT FROM AUTHOR]

Here, we have investigated the possible effect of UV-B light on the folding/unfolding properties and stability of Arabidopsis thaliana MYB4 (AtMYB4) transcription factor in vitro by using biophysical approaches. Urea-induced equilibrium unfolding analyses have shown relatively higher stability of the wild-type recombinant AtMYB4 protein than the N-terminal deletion forms after UV-B exposure. However, as compared to wild-type form, AtMYB4Δ2 protein, lacking both the two N-terminal MYB domains, showed appreciable alteration in the secondary structure following UV-B exposure. UV-B irradiated AtMYB4Δ2 also displayed higher propensity of aggregation in light scattering experiments, indicating importance of the N-terminal modules in regulating the stability of AtMYB4 under UV-B stress. DNA binding assays have indicated specific binding activity of AtMYB4 to a putative MYB4 binding motif located about 212 bp upstream relative to transcription start site of AtMYB4 gene promoter, while relatively weak DNA binding activity was detected for another putative MYB4 motif located at -908 bp in AtMYB4 promoter. Gel shift and fluorescence anisotropy studies have shown increased binding affinity of UV-B exposed AtMYB4 to the promoter proximal MYB4 motif. ChIP assay has revealed binding of AtMYB4 to the promoter proximal (-212 position) MYB4 motif (ACCAAAC) in vivo. Docking experiments further revealed mechanistic detail of AtMYB4 interaction with the putative binding motifs. Overall, our results have indicated that the N-terminal 62–116 amino acid residues constituting the second MYB domain plays an important role in maintaining the stability of the C-terminal region and the overall stability of the protein, while a promoter proximal MYB-motif in AtMYB4 promoter may involve in the regulation of its own expression under UV-B light. [ABSTRACT FROM AUTHOR]

C-to-U editing is an important event in post-transcriptional RNA processing, which converts a specific cytidine (C)-to-uridine (U) in transcripts of mitochondria and plastids. Typically, the pentatricopeptide repeat (PPR) protein, which specifies the target C residue by binding to its upstream sequence, is involved in the editing of one or a few sites. Here we report a novel PPR-DYW protein EMP21 that is associated with editing of 81 sites in maize. EMP21 is localized in mitochondria and loss of the EMP21 function severely inhibits the embryogenesis and endosperm development in maize. From a scan of 35 mitochondrial transcripts produced by the Emp21 loss-of-function mutant, the C-to-U editing was found to be abolished at five sites (nad7-77, atp1-1292, atp8-437, nad3-275 and rps4-870), while reduced at 76 sites in 21 transcripts. In most cases, the failure to editing resulted in the translation of an incorrect residue. In consequence, the mutant became deficient with respect to the assembly and activity of mitochondrial complexes I and V. As six of the decreased editing sites in emp21 overlap with the affected editing sites in emp5-1, and the editing efficiency at rpl16-458 showed a substantial reduction in the emp21-1 emp5-4 double mutant compared with the emp21-1 and emp5-4 single mutants, we explored their interaction. A yeast two hybrid assay suggested that EMP21 does not interact with EMP5, but both EMP21 and EMP5 interact with ZmMORF8. Together, these results indicate that EMP21 is a novel PPR-DYW protein required for the editing of ~17% of mitochondrial target Cs, and the editing process may involve an interaction between EMP21 and ZmMORF8 (and probably other proteins). [ABSTRACT FROM AUTHOR]