SNVstory: inferring genetic ancestry from genome sequencing data.

BACKGROUND: Genetic ancestry, inferred from genomic data, is a quantifiable biological parameter. While much of the human genome is identical across populations, it is estimated that as much as 0.4% of the genome can differ due to ancestry. This variation is primarily characterized by single nucleotide variants (SNVs), which are often unique to specific genetic populations. Knowledge of a patient's genetic ancestry can inform clinical decisions, from genetic testing and health screenings to medication dosages, based on ancestral disease predispositions. Nevertheless, the current reliance on self-reported ancestry can introduce subjectivity and exacerbate health disparities. While genomic sequencing data enables objective determination of a patient's genetic ancestry, existing approaches are limited to ancestry inference at the continental level. RESULTS: To address this challenge, and create an objective, measurable metric of genetic ancestry we present SNVstory, a method built upon three independent machine learning models for accurately inferring the sub-continental ancestry of individuals. We also introduce a novel method for simulating individual samples from aggregate allele frequencies from known populations. SNVstory includes a feature-importance scheme, unique among open-source ancestral tools, which allows the user to track the ancestral signal broadcast by a given gene or locus. We successfully evaluated SNVstory using a clinical exome sequencing dataset, comparing self-reported ethnicity and race to our inferred genetic ancestry, and demonstrate the capability of the algorithm to estimate ancestry from 36 different populations with high accuracy. CONCLUSIONS: SNVstory represents a significant advance in methods to assign genetic ancestry, opening the door to ancestry-informed care. SNVstory, an open-source model, is packaged as a Docker container for enhanced reliability and interoperability. It can be accessed from https://github.com/nch-igm/snvstory .

Elongation Factor P and the Control of Translation Elongation.

Elongation factor P (EF-P) binds to ribosomes requiring assistance with the formation of oligo-prolines. In order for EF-P to associate with paused ribosomes, certain tRNAs with specific d-arm residues must be present in the peptidyl site, e.g., tRNAPro. Once EF-P is accommodated into the ribosome and bound to Pro-tRNAPro, productive synthesis of the peptide bond occurs. The underlying mechanism by which EF-P facilitates this reaction seems to have entropic origins. Maximal activity of EF-P requires a posttranslational modification in Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Each of these modifications is distinct and ligated onto its respective EF-P through entirely convergent means. Here we review the facets of translation elongation that are controlled by EF-P, with a particular focus on the purpose behind the many different modifications of EF-P.

Carbonyl reduction by YmfI in Bacillus subtilis prevents accumulation of an inhibitory EF-P modification state.

Translation elongation factor P (EF-P) in Bacillus subtilis is required for a form of surface migration called swarming motility. Furthermore, B. subtilis EF-P is post-translationally modified with a 5-aminopentanol group but the pathway necessary for the synthesis and ligation of the modification is unknown. Here we determine that the protein YmfI catalyzes the reduction of EF-P-5 aminopentanone to EF-P-5 aminopentanol. In the absence of YmfI, accumulation of 5-aminopentanonated EF-P is inhibitory to swarming motility. Suppressor mutations that enhanced swarming in the absence of YmfI were found at two positions on EF-P, including one that changed the conserved modification site (Lys 32) and abolished post-translational modification. Thus, while modification of EF-P is thought to be essential for EF-P activity, here we show that in some cases it can be dispensable. YmfI is the first protein identified in the pathway leading to EF-P modification in B. subtilis, and B. subtilis encodes the first EF-P ortholog that retains function in the absence of modification.

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P.

Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys(32) of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility.

Non-canonical roles of tRNAs and tRNA mimics in bacterial cell biology.

Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl-tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl-tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non-canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions.

The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation.

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a ß-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The ß-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any other hydroxylase structures reported so far. The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology. Furthermore, YfcM has a metal ion-coordinating motif, similar to YbeY. The metal ion-coordinating motif of YfcM resembles a 2-His-1-carboxylate motif, which coordinates an Fe(II) ion and forms the catalytic site of non-heme iron enzymes. Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the ß-lysylated lysine residue of EF-P. Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.

(R)-ß-lysine-modified elongation factor P functions in translation elongation.

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-ß-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(ß)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the ß-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational ß-lysylation but not hydroxylation.

Amino acid substitutions in human growth hormone affect secondary structure and receptor binding.

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

Multiomics in primary and metastatic breast tumors from the AURORA US network finds microenvironment and epigenetic drivers of metastasis.

The AURORA US Metastasis Project was established with the goal to identify molecular features associated with metastasis. We assayed 55 females with metastatic breast cancer (51 primary cancers and 102 metastases) by RNA sequencing, tumor/germline DNA exome and low-pass whole-genome sequencing and global DNA methylation microarrays. Expression subtype changes were observed in ~30% of samples and were coincident with DNA clonality shifts, especially involving HER2. Downregulation of estrogen receptor (ER)-mediated cell-cell adhesion genes through DNA methylation mechanisms was observed in metastases. Microenvironment differences varied according to tumor subtype; the ER+/luminal subtype had lower fibroblast and endothelial content, while triple-negative breast cancer/basal metastases showed a decrease in B and T cells. In 17% of metastases, DNA hypermethylation and/or focal deletions were identified near HLA-A and were associated with reduced expression and lower immune cell infiltrates, especially in brain and liver metastases. These findings could have implications for treating individuals with metastatic breast cancer with immune- and HER2-targeting therapies.

Identification and characterization of a putative arginine kinase homolog from Myxococcus xanthus required for fruiting body formation and cell differentiation.

Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.