Flaviviruses are emerging human pathogens and worldwide health threats. During infection, a pathogenic, subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5’→3’ host cell exonuclease Xrn1 through an unknown RNA structure-based mechanism. Here, we present the crystal structure of a complete Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly-conserved nucleotides. The RNA’s three-dimensional topology creates a ring-like conformation with the 5’ end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity.

Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3′UTR that interact with Xrn1 to halt processive 5′ to 3′ decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3′ UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3′ UTR architecture, sfRNA production, and host adaptation.

Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition.

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Rheumatoid arthritis is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to patient morbidity. However, the impact of rheumatoid arthritis on hematopoietic stem cells has not been fully elucidated. Using a collagen-induced mouse model of human rheumatoid arthritis, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in hematopoietic stem cells. Surprisingly, despite ongoing inflammation, hematopoietic stem cells from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we find that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra leads to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduces expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in hematopoietic stem cells of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Myelodysplastic syndrome-associated spliceosome gene mutations enhance innate immune signaling Genes encoding spliceosome components including SF3B1, U2AF1, and SRSF2, are frequently somatically mutated in myelodysplastic syndromes (MDS), other hematologic malignancies, and solid tumors. 1 Typically these are mutually exclusive, heterozygous, missense, hotspot mutations that result in neomorphic or gain-offunction splicing phenotypes. These mutations alter splicing of many genes; however, overlap among the different splicing factors is limited. Thus, a common mechanism by which spliceosome mutations contribute to disease is suspected but has remained elusive. We previously demonstrated that inhibiting any of five different spliceosome genes (SF3B1, SF3A1, SF3A2, SF3A3, EFTUD2) in mouse or human macrophages reduces inflammatory cytokine production induced by multiple Toll-like receptor (TLR) agonists including the TLR4 agonist lipopolysaccharide (LPS). 2-5 Although these genes encode essential spliceosome components, partial gene knockdown (approx. 80%) reduces LPS-induced inflammatory cytokine production without affecting viability or phagocytosis. 2-5 Hence, innate immunity may be particularly sensitive to spliceosome perturbation. These observations suggest that MDS-associated spliceosome mutations might enhance innate immunity,

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1β (IL-1β), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell–intrinsic competitive advantage; rather chronic IL-1β exposure engendered potent selection for Cebpa loss. Chronic IL-1β augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1β, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.