Expression Of Paralogs Research Articles

High-resolution reconstruction of a C. elegans ribosome sheds light on evolutionary dynamics and tissue specificity.

Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression and tissue patterning in animals. An invaluable tool in modern gene expression research is the presence of a high-resolution ribosome structure, though no such structure exists for C. elegans Here, we present a high-resolution single-particle cryogenic electron microscopy (cryo-EM) reconstruction and molecular model of a C. elegans ribosome, revealing a significantly streamlined animal ribosome. Many facets of ribosome structure are conserved in C. elegans, including overall ribosomal architecture and the mechanism of cycloheximide, whereas other facets, such as expansion segments and eL28, are rapidly evolving. We identify uL5 and uL23 as two instances of tissue-specific ribosomal protein paralog expression conserved in Caenorhabditis, suggesting that C. elegans ribosomes vary across tissues. The C. elegans ribosome structure will provide a basis for future structural, biochemical, and genetic studies of translation in this important animal system.

Paralog switching facilitates diadromy: ontogenetic, microevolutionary and macroevolutionary evidence.

Identifying how the demands of migration are met at the level of gene expression is critical for understanding migratory physiology and can potentially reveal how migratory forms evolve from nonmigratory forms and vice versa. Among fishes, migration between freshwater and seawater (diadromy) requires considerable osmoregulatory adjustments, powered by the ion pump Na+, K+-ATPase (NKA) in the gills. Paralogs of the catalytic α-subunit of the pump (NKA α1a and α1b) are reciprocally upregulated in fresh- and seawater, a response known as paralog-switching, in gills of some diadromous species. We tested ontogenetic changes in NKA α-subunit paralog expression patterns, comparing pre-migrant and migrant alewife (Alosa pseudoharengus) sampled in their natal freshwater environment and after 24h in seawater. In comparison to pre-migrants, juvenile out-migrants exhibited stronger paralog switching via greater downregulation of NKA α1a in seawater. We also tested microevolutionary changes in the response, exposing juvenile diadromous and landlocked alewife to freshwater (0 ppt) and seawater (30 ppt) for 2, 5, and 15days. Diadromous and landlocked alewife exhibited salinity-dependent paralog switching, but levels of NKA α1b transcription were higher and the decrease in NKA α1a was greater after seawater exposure in diadromous alewife. Finally, we placed alewife α-subunit NKA paralogs in a macroevolutionary context. Molecular phylogenies show alewife paralogs originated independently of paralogs in salmonids and other teleosts. This study demonstrated that NKA paralog switching is tied to halohabitat profile and that duplications of the NKA gene provided the substrate for multiple, independent molecular solutions that support a diadromous life history.

Leishmania Ribosomal Protein (RP) paralogous genes compensate each other's expression maintaining protein native levels.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3'UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3'UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Cartography of teneurin and latrophilin expression reveals spatiotemporal axis heterogeneity in the mouse hippocampus during development.

Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.

Duplicate Gene Expression and Possible Mechanisms of Paralog Retention During Bacterial Genome Expansion.

Gene duplication contributes to the evolution of expression and the origin of new genes, but the relative importance of different patterns of duplicate gene expression and mechanisms of retention remains debated and particularly poorly understood in bacteria. Here, we investigated gene expression patterns for two lab strains of the cyanobacterium Acaryochloris marina with expanding genomes that contain about 10-fold more gene duplicates compared with most bacteria. Strikingly, we observed a generally stoichiometric pattern of greater combined duplicate transcript dosage with increased gene copy number, in contrast to the prevalence of expression reduction reported for many eukaryotes. We conclude that increased transcript dosage is likely an important mechanism of initial duplicate retention in these bacteria and may persist over long periods of evolutionary time. However, we also observed that paralog expression can diverge rapidly, including possible functional partitioning, for which different copies were respectively more highly expressed in at least one condition. Divergence may be promoted by the physical separation of most Acaryochloris duplicates on different genetic elements. In addition, expression pattern for ancestrally shared duplicates could differ between strains, emphasizing that duplicate expression fate need not be deterministic. We further observed evidence for context-dependent transcript dosage, where the aggregate expression of duplicates was either greater or lower than their single-copy homolog depending on physiological state. Finally, we illustrate how these different expression patterns of duplicated genes impact Acaryochloris biology for the innovation of a novel light-harvesting apparatus and for the regulation of recA paralogs in response to environmental change.

Role of paralogs in the sex-bias transcriptional and metabolic regulation of the brain-placental axis in mice

IntroductionDuplicated genes or paralogs play important roles in the adaptive function of eukaryotic genomes. Animal studies have shown evidence for the functional role of paralogs in pregnancy, but our knowledge about the role of paralogs in the fetoplacental regulation remains limited. In particular, if fetoplacental metabolic regulation is modulated by differential expression of paralogs remains unexamined. MethodsIn this study, gene expression profiles of day-15 placenta and fetal brain were compared to identify families or groups of paralogous genes expressed in the placenta and brain of male versus female fetuses in mice. A Bayesian modeling was applied to infer directional relationship of transcriptional variation of the paralogs relative to the phylogenetic variation of the genes in each family. Gas chromatography-mass spectrometry (GC-MS) was used to perform untargeted metabolomics analysis of day-15 placenta and fetal brain of both sexes. ResultsWe identified paralog groups that were expressed in a sex and/or tissue biased manner between the placenta and fetal brain. Bayesian modeling showed evidence for directional relationship between expression and phylogeny of specific paralogs. These relationships were sex specific. GC-MS analysis identified metabolites that were expressed in a sex-bias manner between the placenta and fetal brain. By performing integrative analysis of the metabolomics and gene expression data, we showed that specific groups of metabolites and paralogous genes were expressed in a coordinated manner between the placenta and fetal brain. DiscussionThe findings of this study collectively suggest that paralogs play an influential role in the regulation of the brain-placental axis in mice.

Active compensation for changes in TDH3 expression mediated by direct regulators of TDH3 in Saccharomyces cerevisiae.

Genetic networks are surprisingly robust to perturbations caused by new mutations. This robustness is conferred in part by compensation for loss of a gene's activity by genes with overlapping functions, such as paralogs. Compensation occurs passively when the normal activity of one paralog can compensate for the loss of the other, or actively when a change in one paralog's expression, localization, or activity is required to compensate for loss of the other. The mechanisms of active compensation remain poorly understood in most cases. Here we investigate active compensation for the loss or reduction in expression of the Saccharomyces cerevisiae gene TDH3 by its paralog TDH2. TDH2 is upregulated in a dose-dependent manner in response to reductions in TDH3 by a mechanism requiring the shared transcriptional regulators Gcr1p and Rap1p. TDH1, a second and more distantly related paralog of TDH3, has diverged in its regulation and is upregulated by another mechanism. Other glycolytic genes regulated by Rap1p and Gcr1p show changes in expression similar to TDH2, suggesting that the active compensation by TDH3 paralogs is part of a broader homeostatic response mediated by shared transcriptional regulators.

Paralog transcriptional differentiation in the D. melanogaster-specific gene family Sdic across populations and spermatogenesis stages

How recently originated gene copies become stable genomic components remains uncertain as high sequence similarity of young duplicates precludes their functional characterization. The tandem multigene family Sdic is specific to Drosophila melanogaster and has been annotated across multiple reference-quality genome assemblies. Here we show the existence of a positive correlation between Sdic copy number and total expression, plus vast intrastrain differences in mRNA abundance among paralogs, using RNA-sequencing from testis of four strains with variable paralog composition. Single cell and nucleus RNA-sequencing data expose paralog expression differentiation in meiotic cell types within testis from third instar larva and adults. Additional RNA-sequencing across synthetic strains only differing in their Y chromosomes reveal a tissue-dependent trans-regulatory effect on Sdic: upregulation in testis and downregulation in male accessory gland. By leveraging paralog-specific expression information from tissue- and cell-specific data, our results elucidate the intraspecific functional diversification of a recently expanded tandem gene family.

Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy.

Identification of synthetic lethal genes with KRASG12C using genome-wide CRISPR/Cas9 screening and credentialing of the ability of TEAD inhibition to enhance KRASG12C efficacy provides a roadmap for combination strategies. See related commentary by Johnson and Haigis, p. 4005.

New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida

Several R2R3-MYB genes control anthocyanin pigmentation in petunia, and ANTHOCYANIN-2 (AN2) is treated as the main player in petal limbs. However, the actual roles of R2R3-MYBs in the coloration of different floral tissues in the so called “darkly-veined” petunias are still not clear. The genetic background and expression of AN2 paralogs from various petunias with different color patterns were identified. All “darkly-veined” genotypes have the identical mutation in the AN2 gene, but express a different functional paralog – ANTHOCYANIN-4 (AN4) – abundantly in flowers. Constitutive overexpression of PhAN4 in this petunia resulted not only in a fully colored flower but also in a clearly visible pigmentation in the green tissue and roots, which can be rapidly increased by stress conditions. Suppression of AN4 gene resulted in discolored petals and whitish anthers. Interestingly, when a similar white flower phenotype was achieved by knockout of an essential structural gene of anthocyanin biosynthesis – CHALCONE ISOMERASE-A (CHI-A) – the plant responded directly by upregulating of another paralogs – DEEP PURPLE (DPL) and PURPLE HAZE (PHZ). Moreover, we also found that CHI–B can partially substitute for CHI-A in anthers, but not in vegetative tissues. Further, no significant effects on the longevity of white or enhanced colored flowers were observed compared with the wild type. We concluded that endogenous up-regulation of AN4 leads to the restoration of petal color in the “darkly-veined” phenotypes as a result of the breeding process under human selection, and CHI–B is a backup for CHI-A acitvity in some floral tissues.

Diversity in Cell Morphology, Composition, and Function among Adipose Depots in River Buffaloes.

Fat deposition is a significant economic trait in livestock animals. Adipose tissues (ATs) developed in subcutaneous and visceral depots are considered waste whereas those within muscle are highly valued. In river buffaloes, lipogenesis is highly active in subcutaneous (especially in the sternum subcutaneous) and visceral depots but not in muscle tissue. Revealing the features and functions of ATs in different depots is significant for the regulation of their development. Here, we characterize the cell size, composition, and function of six AT depots in river buffaloes. Our data support that the subcutaneous AT depots have a larger cell size than visceral AT depots, and the subcutaneous AT depots, especially the sternum subcutaneous AT, are mainly associated with the extracellular matrix whereas the visceral AT depots are mainly associated with immunity. We found that sternum subcutaneous AT is significantly different from ATs in other depots, due to the high unsaturated fatty acid content and the significant association with metabolic protection. The perirenal AT is more active in FA oxidation for energy supply. In addition, the expression of HOX paralogs supports the variable origins of ATs in different depots, indicating that the development of ATs in different depots is mediated by their progenitor cells. The present study enhances our understanding of the cellular and molecular features, metabolism, and origin of AT depots in buffaloes, which is significant for the regulation of fat deposition and provides new insights into the features of AT depots in multiple discrete locations.

Fast and slow skeletal myosin binding protein-C and aging

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs.

Shared cis-regulatory modules control expression of the tandem paralogs midline and H15 in the follicular epithelium.

The posterior end of the follicular epithelium is patterned by midline (MID) and its paralog H15, the Drosophila homologs of the mammalian Tbx20 transcription factor. We have previously identified two cis-regulatory modules (CRMs) that recapitulate the endogenous pattern of mid in the follicular epithelium. Here, using CRISPR/Cas9 genome editing, we demonstrate redundant activity of these mid CRMs. Although the deletion of either CRM alone generated marginal change in mid expression, the deletion of both CRMs reduced expression by 60%. Unexpectedly, the deletion of the 5' proximal CRM of mid eliminated H15 expression. Interestingly, expression of these paralogs in other tissues remained unaffected in the CRM deletion backgrounds. These results suggest that the paralogs are regulated by a shared CRM that coordinates gene expression during posterior fate determination. The consistent overlapping expression of mid and H15 in various tissues may indicate that the paralogs could also be under shared regulation by other CRMs in these tissues.

IRAK1 Contributes to IRAK4 Inhibitor Resistance Via Non-Canonical Signaling Mechanisms in MDS/AML

IRAKs are a family of related kinases that operate at the nexus of multiple innate immune and inflammatory pathways implicated in myeloid malignancies. IRAK4 inhibitors have advanced into clinical trials for MDS and AML validating IRAK4 as a therapeutic target. Early data from these trials has been encouraging; however, the overall responses remain modest. Herein, we explored mechanisms of resistance to IRAK4-selective inhibitors in MDS/AML. Our findings uncovered non-canonical IRAK1/4 signaling paradigms driving leukemic stem and progenitor cells (LSPC) and yielded novel therapeutic strategies for MDS/AML. Consistent with the initial observations from the clinical trials, inhibition or deletion of IRAK4 in a panel of MDS/AML cell lines and patient samples resulted in incomplete suppression of LSPCs and a corresponding activation of innate immune pathways. Given the evolutionary conserved redundancy of IRAK-dependent pathways, we first examined the expression of IRAK paralogs. In IRAK4 knockout (KO) MDS/AML cells, we only observed overexpression and activation of the IRAK4 paralog, IRAK1. IRAK4 kinase inhibitors or PROTACs resulted in a similar compensatory activation of IRAK1 in MDS/AML. To validate the compensation of IRAK1, we deleted IRAK1 in MDS/AML cells and found that concomitant suppression of IRAK1 led to a significant reduction of LSPC function in IRAK4-KO MDS/AML cells. Co-deletion of IRAK1 and IRAK4 similarly reduced leukemic engraftment and extended survival in an AML xenograft model. Hence, IRAK1 mitigates the efficacy of IRAK4-inhibitors in MDS/AML. Canonical IRAK signaling depends on recruitment of MyD88 and IRAK4 to activated receptors, which results in the subsequent recruitment and activation of IRAK1. Based on this canonical model, IRAK1 and IRAK4 are thus independently essential for signaling. Since dual inhibition of IRAK1/4 is obligatory for suppression of LSPCs, we posited that conventional IRAK signaling is not sufficient nor essential in MDS/AML. Therefore, we first examined the role of proximal upstream (MyD88) and downstream (TRAF6) effectors of IRAK1/4. Deletion of TRAF6 resulted in suppression of LSPCs as observed upon inhibition of IRAK1/4. In contrast, MyD88-KO AML cells did not exhibit a functional defect but did retain sensitivity to deletion of IRAK1 and IRAK4, indicating canonical MyD88-dependent signaling is not operational in MDS/AML. To investigate non-canonical IRAK1/4 signaling we performed RNA- and ATAC-seq and identified gene expression programs dependent on IRAK1 and/or IRAK4, yet independent of MyD88. IRAK1/4 deletion correlated with dysregulation of transcription factors involved in stem cell maintenance and myeloid differentiation. The requirement for IRAK1/4 in preserving an undifferentiated LSPC state was corroborated by morphological assessment of IRAK1/4-KO MDS/AML cells. To delineate the signaling mechanisms by which IRAK1/4 governs stem programs, we performed mass spectrometry proteomics and identified unique IRAK4- and IRAK1-interacting proteins. Integration of the proteomic and transcriptomic studies identified pathways (HIF1A, STAT3/IRFs, E2F4) implicated in stem cell and undifferentiated states as effectors of IRAK1 and IRAK4. To extend these studies to improve the efficacy of IRAK4-targeted therapy in MDS/AML, we developed a novel series of structurally related inhibitors targeting IRAK1 and IRAK4 (KME-2780: IRAK1 IC50 = 32 nM; IRAK4 IC50 = 0.9 nM) or IRAK4 alone (KME-3859: IRAK1 IC50 = >500 nM; IRAK4 IC50 = 5 nM). In human AML cell lines and patient samples, the dual IRAK1/4 inhibitor was significantly more effective at inducing cell death and differentiation, and attenuating LSPC function as compared to the IRAK4 inhibitor. Thus, the mandate for targeting both IRAK1 and IRAK4 established in our genetic studies translates to pharmacologic interventions. Overall, we demonstrate that compensation by IRAK1 is a barrier to IRAK4-directed therapy and reveal that dual IRAK1/4 inhibitors are needed for achieving optimal clinical response in MDS/AML. In the process, we also uncovered novel and non-canonical signaling paradigms governing the oncogenic role of IRAK1/4.

Variable paralog expression underlies phenotype variation.

Human faces are variable; we look different from one another. Craniofacial disorders further increase facial variation. To understand craniofacial variation and how it can be buffered, we analyzed the zebrafish mef2ca mutant. When this transcription factor encoding gene is mutated, zebrafish develop dramatically variable craniofacial phenotypes. Years of selective breeding for low and high penetrance of mutant phenotypes produced strains that are either resilient or sensitive to the mef2ca mutation. Here, we compared gene expression between these strains, which revealed that selective breeding enriched for high and low mef2ca paralog expression in the low- and high-penetrance strains, respectively. We found that mef2ca paralog expression is variable in unselected wild-type zebrafish, motivating the hypothesis that heritable variation in paralog expression underlies mutant phenotype severity and variation. In support, mutagenizing the mef2ca paralogs, mef2aa, mef2b, mef2cb, and mef2d demonstrated modular buffering by paralogs. Specifically, some paralogs buffer severity while others buffer variability. We present a novel, mechanistic model for phenotypic variation where variable, vestigial paralog expression buffers development. These studies are a major step forward in understanding the mechanisms of facial variation, including how some genetically resilient individuals can overcome a deleterious mutation.

Origins, genomic structure and copy number variation of snake venom myotoxins

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous β-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

Social status-dependent regulation and function of the somatotropic axis in juvenile rainbow trout

Juvenile rainbow trout (Oncorhynchus mykiss) develop social hierarchies when competing for resources in a constrained environment. Among the physiological consequences of social status are changes in organismal energy metabolism, which generally favour anabolic pathways in dominant fish and catabolic pathways in subordinate fish. The somatotropic axis is an important regulator of metabolism and growth that could be involved in mediating metabolic changes in response to social status in juvenile rainbow trout. Here we used juvenile trout housed either in dyads or individually (sham controls) to determine whether social status changes indices of somatotropic axis function. Although pituitary growth hormone expression (gh1 and gh2) did not differ among groups, circulating growth hormone (GH) increased ∼12-fold in subordinate fish compared to sham and dominant fish. Social status caused consistent differential expression of GH receptor paralogues in liver and muscle, two principal target tissues of GH. Compared to dominant and/or sham fish, ghra paralogue expression (ghra1 and ghra2) was lower, while ghrb1 expression was higher in subordinate fish. Across tissues, ghra paralogue expression was generally positively correlated with expression of insulin growth factors (igf1, igf2), while ghrb1 expression was positively correlated with transcript abundance of hormone sensitive lipase (hsl1). Because igf and hsl expression are subject to context-dependent GH control in rainbow trout, these results suggest that increased circulating GH in conjunction with differential expression of ghr paralogues may translate into prioritization of downstream catabolic lipolytic pathways in subordinate rainbow trout. These findings support a social context-dependent role for GH signalling in mediating metabolic changes in juvenile rainbow trout.

Allele-aware chromosome-level genome assembly of Artemisia annua reveals the correlation between ADS expansion and artemisinin yield

Artemisia annua is the major natural source of artemisinin, an anti-malarial medicine commonly used worldwide. Here, we present chromosome-level haploid maps for two A. annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production. High-fidelity sequencing, optical mapping, and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A. annua. Approximately 50,000 genes were annotated for each haplotype genome, and a triplication event that occurred approximately 58.12 million years ago was examined for the first time in this species. A total of 3,903,467–5,193,414 variants (SNPs, indels, and structural variants) were identified in the 1.5-Gb genome during pairwise comparison between haplotypes, consistent with the high heterozygosity of this species. Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-diene synthase genes. This correlation was further confirmed by resequencing of 36 A. annua samples with varied artemisinin contents. Circular consensus sequencing of transcripts facilitated the detection of paralog expression. Collectively, our study provides chromosome-level allele-aware genome assemblies for two A. annua strains and new insights into the biosynthesis of artemisinin and its regulation, which will contribute to conquering malaria worldwide.

Conserved exchange of paralog proteins during neuronal differentiation.

Gene duplication enables the emergence of new functions by lowering the evolutionary pressure that is posed on the ancestral genes. Previous studies have highlighted the role of specific paralog genes during cell differentiation, for example, in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. Whereas ∼80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B members of the COPII complex. Altering the ratio between these two genes via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.

Gene family evolution underlies cell-type diversification in the hypothalamus of teleosts.

Hundreds of cell types form the vertebrate brain but it is largely unknown how similar cellular repertoires are between or within species or how cell-type diversity evolves. To examine cell-type diversity across and within species, we performed single-cell RNA sequencing of ~130,000 hypothalamic cells from zebrafish (Danio rerio) and surface and cave morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which diverged from a common ancestor over 150 million years ago. Shared cell types displayed shifts in paralogue expression that were generated by subfunctionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes and characterized by the neofunctionalization of expression patterns of members of recently expanded or contracted gene families. Comparisons between surface and cave morphs revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types and reveal how gene family evolution and shifts in paralogue expression contribute to cellular diversity.