RNA binding protein DDX5 restricts RORγt+ Treg suppressor function to promote intestine inflammation.

Retinoid-related orphan receptor (RAR) gamma (RORγt)-expressing regulatory T cells (RORγt+ Tregs) play pivotal roles in preventing T cell hyperactivation and maintaining tissue homeostasis, in part by secreting the anti-inflammation cytokine interleukin-10 (IL-10). Here, we report that hypoxia-induced factor 1α (HIF1α) is the master transcription factor for Il10 in RORγt+ Tregs. This critical anti-inflammatory pathway is negatively regulated by an RNA binding protein DEAD box helicase 5 (DDX5). As a transcriptional corepressor, DDX5 restricts the expression of HIF1α and its downstream target gene Il10 in RORγt+ Tregs. T cell-specific Ddx5 knockout (DDX5ΔT) mice have augmented RORγt+ Treg suppressor activities and are better protected from intestinal inflammation. Genetic ablation or pharmacologic inhibition of HIF1α restores enteropathy susceptibility in DDX5ΔT mice. The DDX5-HIF1α-IL-10 pathway is conserved in mice and humans. These findings reveal potential therapeutic targets for intestinal inflammatory diseases.

RORγt phosphorylation protects against T cell-mediated inflammation.

RAR-related orphan receptor-γ (RORγt) is an essential transcription factor for thymic T cell development, secondary lymphoid tissue organogenesis, and peripheral immune cell differentiation. Serine 182 phosphorylation is a major post-translational modification (PTM) on RORγt. However, the in vivo contribution of this PTM in health and disease settings is unclear. We report that this PTM is not involved in thymic T cell development and effector T cell differentiation. Instead, it is a critical regulator of inflammation downstream of IL-1ß signaling and extracellular signal regulated kinases (ERKs) activation. ERKs phosphorylation of serine 182 on RORγt serves to simultaneously restrict Th17 hyperactivation and promote anti-inflammatory cytokine IL-10 production in RORγt+ Treg cells. Phospho-null RORγtS182A knockin mice experience exacerbated inflammation in models of colitis and experimental autoimmune encephalomyelitis (EAE). In summary, the IL-1ß-ERK-RORγtS182 circuit protects against T cell-mediated inflammation and provides potential therapeutic targets to combat autoimmune diseases.

RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine.

OBJECTIVE: Tuft cells residing in the intestinal epithelium have diverse functions. In the small intestine, they provide protection against inflammation, combat against helminth and protist infections, and serve as entry portals for enteroviruses. In the colon, they had been implicated in tumourigenesis. Commitment of intestinal progenitor cells to the tuft cell lineage requires Rho GTPase Cell Division Cycle 42 (CDC42), a Rho GTPase that acts downstream of the epidermal growth factor receptor and wingless-related integration site signalling cascades, and the master transcription factor POU class 2 homeobox 3 (POU2F3). This study investigates how this pathway is regulated by the DEAD box containing RNA binding protein DDX5 in vivo. DESIGN: We assessed the role of DDX5 in tuft cell specification and function in control and epithelial cell-specific Ddx5 knockout mice (DDX5ΔIEC) using transcriptomic approaches. RESULTS: DDX5ΔIEC mice harboured a loss of intestinal tuft cell populations, modified microbial repertoire, and altered susceptibilities to ileal inflammation and colonic tumourigenesis. Mechanistically, DDX5 promotes CDC42 protein synthesis through a post-transcriptional mechanism to license tuft cell specification. Importantly, the DDX5-CDC42 axis is parallel but distinct from the known interleukin-13 circuit implicated in tuft cell hyperplasia, and both pathways augment Pou2f3 expression in secretory lineage progenitors. In mature tuft cells, DDX5 not only promotes integrin signalling and microbial responses, it also represses gene programmes involved in membrane transport and lipid metabolism. CONCLUSION: RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine.

DDX5 promotes oncogene C3 and FABP1 expressions and drives intestinal inflammation and tumorigenesis.

Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid-binding protein 1 (FABP1). However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that the RNA-binding protein DDX5 binds to the mRNA transcripts of C3 and Fabp1 to augment their expressions posttranscriptionally. Knocking out DDX5 in epithelial cells protected mice from intestinal tumorigenesis and dextran sodium sulfate (DSS)-induced colitis. Identification of DDX5 as a common upstream regulator of tissue-specific oncogenic molecules provides an excellent therapeutic target for intestinal diseases.

A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses.

The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here, we report the generation of over 14000 artificial microRNA (amiRNA)-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol was developed for isolating robust and reproducible amiRNA mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA-insensitive amiRNA line that targets three avirulence-induced gene 2(-like) genes was isolated . A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led to the isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 11000 T2 amiRNA lines (with 3000 lines in progress) generated here provides a new platform for forward genetic screens and has been made available to the Arabidopsis Biological Resource Center (ABRC). Optimized procedures for amiRNA screening and controls are described.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

Bruno-like proteins modulate flowering time via 3' UTR-dependent decay of SOC1 mRNA.

The Bruno RNA-binding protein (RBP) has been shown to initially repress the translation of oskar mRNA during Drosophila oogenesis and later to be involved in a broad range of RNA regulation. Here, we show that homologous constitutive overexpression of each of two Arabidopsis thaliana Bruno-like genes, AtBRN1 and AtBRN2, delayed the flowering time, while the atbrn1 atbrn2-3 double mutant flowered early and exhibited increased expression of APETALA1 (AP1) and LEAFY (LFY) transcripts. Crossing of 35S::AtBRNs with SOC1 101-D plants demonstrated that 35S::AtBRNs suppress an early-flowering phenotype of SOC1 101-D in which the coding sequence (CDS) with the 3' UTR of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) gene is overexpressed. However, this early-flowering phenotype by SOC1 overexpression was maintained in the plants coexpressing 35S::AtBRNs and 35S::SOC1 without the 3' UTR (-3' UTR). Using yeast three-hybrid, electrophoretic mobility shift, RNA immunoprecipitation, and protoplast transient assays, we found that AtBRNs bind to the 3' UTR of SOC1 RNA and participate in mRNA decay, which was mediated by the distal region of the SOC1 3' UTR. Overall, AtBRNs repress SOC1 activity in a 3' UTR-dependent manner, thereby controlling the flowering time in Arabidopsis.

Pumilio Puf domain RNA-binding proteins in Arabidopsis.

Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35-39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example.

APUM23, a nucleolar Puf domain protein, is involved in pre-ribosomal RNA processing and normal growth patterning in Arabidopsis.

Pumilio, an RNA-binding protein that contains tandemly repeated Puf domains, is known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. APUM23 is constitutively expressed, with higher levels in metabolically active tissues, and its expression is up-regulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth, with serrated and scrunched leaves, an abnormal venation pattern, and distorted organization of the palisade parenchyma cells - a phenotype that is reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on this localization, rRNA processing was examined. In apum23, 35S pre-rRNA, and unprocessed 18S and 5.8S poly(A) rRNAs, accumulated without affecting the steady-state levels of mature rRNAs, indicating that APUM23 is involved in the processing and/or degradation of 35S pre-rRNA and rRNA maturation by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 small nucleolar RNAs (snoRNAs) and accumulated RNAs within the nucleolus. Our data suggest that APUM23 plays an important role in plant development via rRNA processing.

The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis.

Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxial-abaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5'-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxial-abaxial polarity.