Etsuko N. Moriyama

Transfer RNA-derived small RNAs (tDRs) represent a diverse class of noncoding RNAs with emerging regulatory roles across all domains of life. In the green alga Chlamydomonas reinhardtii, next-generation sequencing revealed abundant, specific tDRs, with a predominance of those derived from plastid-encoded transfer RNAs (tRNAs). Most of these tDRs map to the 5′ ends of mature tRNAs and are 29 to 33 nucleotides in length, suggesting that they arise from cleavage at the anticodon loop or stem. We confirmed the plastid origin of some tDRs by engineering the chloroplast genome to replace the plastid-encoded tRNAAsn(GTT) gene with a nuclear counterpart. As reported in land plants, DICER-LIKE endonucleases play a limited role in tDR biogenesis in Chlamydomonas. However, a distinct subset of abundant tDRs, originating from both nucleus- and chloroplast-encoded tRNAs, associates with ARGONAUTE1 (AGO1). Using Gaussia luciferase reporter assays, we demonstrated that AGO1-associated tDRs can mediate RNAi-like gene silencing and that tDRs originating from plastid-encoded tRNAs can function outside the chloroplast. Additionally, the tDR population in Chlamydomonas undergoes dynamic changes in response to nutrient availability. While nitrogen starvation causes a general decrease in both mature tRNA and tDR levels, sulfur deprivation specifically induces the accumulation of certain tDRs. Furthermore, strains in which the plastid tRNAAsn(GTT) gene was replaced by a nuclear counterpart exhibited phenotypic defects under sulfur-deprived conditions, suggesting a potential regulatory role for plastid-derived tDRs in nutrient stress responses. Collectively, these findings expand our understanding of tDR functions, particularly those of chloroplast origin, in photosynthetic eukaryotes.

The emergence of jaws in early vertebrates introduced a novel feeding apparatus and powerful oral defenses, but it also increased the risk of physical injury and pathogen exposure. Interferon regulatory factors (IRFs) play critical roles in orchestrating innate immunity and inflammation in response to invading microbes and tissue damage, with their subcellular localization being essential to some IRFs' function. Our results indicate that IRF members underwent independent expansion and diversification in two distinct vertebrate lineages: jawed and jawless vertebrates. The jawed vertebrate‐specific factor, IRF5, has maintained conserved nuclear export sequences throughout evolution, while newly diversified IRF members in jawed vertebrates have acquired cytoplasmic localization. This cytoplasmic shift particularly affected IRFs involved in type I interferon (IFN) signaling (IRF3, IRF5, IRF7, and IRF9), suggesting co‐evolution with the development of the type I IFN system in jawed animals. Interestingly, although IRF9 is inherently nuclear, its association with Signal Transducer and Activator of Transcription 2 (STAT2) has led to its cytoplasmic localization. Additionally, IRF6, another jawed vertebrate‐specific factor, plays a crucial role in jaw development, reflecting an evolutionary adaptation that aligns structural innovations with immune function. Our findings suggest that the evolution of jaws coincided with the adoption of cytoplasmic localization in IRF members, potentially enhancing rapid immune responses to meet the immunological challenges posed by the predatory lifestyle of early jawed vertebrates.

The emergence of jaws in early vertebrates introduced a novel feeding apparatus and potent oral defenses but also increased the risk of physical injury and pathogen exposure. Innate immunity and inflammation constitute the body’s first line of defense against invading microbes and tissue damage, aiming to eliminate threats and restore internal homeostasis. Interferon regulatory factor 5 (IRF5) plays a critical role in orchestrating innate immunity and inflammation by regulating the transcription of genes that encode type I interferons (IFNs) and pro-inflammatory cytokines. Despite this, the evolution of IRF5 has remained poorly understood. We have identified the IRF5 and IRF6 genes in cartilaginous fish, including sharks. As cartilaginous fish represent one of the oldest surviving jawed vertebrate lineages, the presence of these genes suggests the genes have ancient origins potentially dating back hundreds of millions of years to early jawed vertebrates. Furthermore, our analysis shows that IRF5 has conserved nuclear export sequences and phosphorylation sites for activation throughout evolution from cartilaginous fish to humans, indicating these regulatory elements evolved early and have been maintained across jawed vertebrates. Additionally, the shift in subcellular localization of IRF5 from nucleus to cytosol, and of other interferon related IRFs, aligns with functional enhancements of IRFs in innate immunity and the emergence of IFNs across jawed vertebrates. This analysis implies that the evolution of jaws may have driven the emergence of new IRF members, the expansion of their functions, and the development of a unique inflammation and innate immune system.

Interferon regulatory factor 5 (IRF5) is a critical transcription factor, regulating the production of inflammatory cytokines and interferons. Dysregulation of IRF5 has been linked to various autoimmune and inflammatory diseases. Pseudogenes may exhibit gene regulatory functions in various mechanisms. In this study, we find the human IRF5 pseudogene 1 (IRF5P1) is a chimeric processed pseudogene containing sequences derived from multiple sources. Remarkably, IRF5P1 is specific to higher primates such as apes and humans. We propose that IRF5P1 may have originated through the ancient integration of a retroviral sequence containing IRF5 mRNA from other animals. Interestingly, IRF5P1 overlaps with another gene, triple QxxK/R motif containing (TRIQK), and the antisense strand of IRF5P1 is predominantly expressed, likely as part of the TRIQK pre-mRNA. The antisense IRF5P1 RNA may regulate IRF5 expression through complementary binding to the IRF5 mRNA, and variants in the IRF5 gene may modulate this interaction. Our analysis suggests that IRF5P1 likely had positive effects on primate evolution, making IRF5P1 a favored component to maintain in the primate lineage, and rendering the pseudogene primate specific. Further studies are essential to elucidate the potential for IRF5P1 to participate in regulatory networks governing IRF5 activity and inflammation and innate immunity.

Small RNAs (sRNAs) associate with ARGONAUTE (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including 3 AGOs and 3 DICER-like proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long (>26 nt) sRNAs that associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these >26 nt sRNAs are derived from moderately repetitive genomic clusters and their biogenesis is DICER-independent. Interestingly, the sequences generating these >26-nt sRNAs have been conserved and amplified in several Chlamydomonas species. Moreover, expression of these longer sRNAs increases substantially under nitrogen or sulfur deprivation, concurrently with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences from which >26-nt sRNAs are produced might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, certain sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

Salmonella enterica is, globally, an important cause of human illness with beef being a significant attributable source. In the human patient, systemic Salmonella infection requires antibiotic therapy, and when strains are multidrug resistant (MDR), no effective treatment may be available. MDR in bacteria is often associated with the presence of mobile genetic elements (MGE) that mediate horizontal spread of antimicrobial resistance (AMR) genes. In this study, we sought to determine the potential relationship of MDR in bovine Salmonella isolates with MGE. The present study involved 111 bovine Salmonella isolates obtained collectively from specimens derived from healthy cattle or their environments at Midwestern U.S. feedyards (2000–2001, n = 19), or specimens from sick cattle submitted to the Nebraska Veterinary Diagnostic Center (2010–2020, n = 92). Phenotypically, 33/111 isolates (29.7%) were MDR (resistant to ≥3 drug classes). Based on whole-genome sequencing (WGS; n = 41) and PCR (n = 111), a MDR phenotype was strongly associated (OR = 186; p < 0.0001) with carriage of ISVsa3, an IS91-like Family transposase. In all 41 isolates analyzed by WGS ((31 MDR and 10 non-MDR (resistant to 0–2 antibiotic classes)), MDR genes were associated with carriage of ISVsa3, most often on an IncC type plasmid carrying bla_CMY-2. The typical arrangement was floR, tet(A), aph(6)-Id, aph(3″)-Ib, and sul2 flanked by ISVsa3. These results suggest that AMR genes in MDR S. enterica isolates of cattle are frequently associated with ISVsa3 and carried on IncC plasmids. Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella strains.

The binding of interferon (IFN) to its receptors leads to formation of IFN‐stimulated gene factor 3 (ISGF3) complex that activates the transcription of cellular IFN‐regulated genes. IFN regulatory factor 9 (IRF9, also called ISGF3γ or p48) is a key component of ISGF3. However, there is limited knowledge regarding the molecular evolution of IRF9 among vertebrates. In this study, we have identified the existence of the IRF9 gene in cartilaginous fish (sharks). Among primates, several isoforms unique to old world moneys and great apes are identified. These IRF9 isoforms are named as primate‐specific IRF9 (PS‐IRF9) to distinguish from canonical IRF9. PS‐IRF9 originates from a unique exon usage and differential splicing in the IRF9 gene. Although the N‐terminus are identical for all IRF9s, the C‐terminal regions of the PS‐IRF9 are completely different from canonical IRF9. In humans, two PS‐IRF9s are identified and their RNA transcripts were detected in human primary peripheral blood mononuclear cells. In addition, human PS‐IRF9 proteins were detected in human cell lines. Sharing the N‐terminal exons with the canonical IRF9 proteins, PS‐IRF9 is predicted to bind to the same DNA sequences as the canonical IRF9 proteins. As the C‐terminal regions of IRFs are the determinants of IRF functions, PS‐IRF9 may offer unique biological functions and represent a novel signaling molecule involved in the regulation of the IFN pathway in a primate‐specific manner.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia , closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including three AGOs and three Dicer-like (DCL) proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long sRNAs (>26 nt), which associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these long sRNAs are derived from moderately repetitive genomic clusters and their biogenesis appears to be Dicer-independent. Interestingly, long sRNA encoding sequences have been conserved and amplified in phylogenetically related Chlamydomonas species. Additionally, expression of several long sRNAs increases substantially under nutrient deprivation, correlating with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences encoding long sRNAs might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, some long sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.