The RNA-binding landscape of HAX1 protein indicates its involvement in ribosome biogenesis and translation.

HAX1 is a human protein with no known homologues or structural domains, mutations in which cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here we report transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in ribosome biogenesis and rRNA processing. Using CRISPR knockouts we find that RNA targets of HAX1 partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. Functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

Genome-Wide DNA Methylome and Transcriptome Analysis of Porcine Testicular Cells Infected With Transmissible Gastroenteritis Virus

Transmissible gastroenteritis virus (TGEV) is a porcine pathogen causing highly communicable gastrointestinal infection that are lethal for suckling piglets. In an attempt to delineate the pathogenic mechanism of TGEV-infected porcine testicular cells (ST cells), we conducted a whole genome analysis of DNA methylation and expression in ST cells through reduced bisulfate-seq and RNA-seq. We examined alterations in the methylation patterns and recognized 1764 distinct methylation sites. 385 differentially expressed genes (DEGs) were enriched in the viral defense and ribosome biogenesis pathways. Integrative analysis identified two crucial genes (EMILIN2, RIPOR3), these two genes expression were negatively correlated to promoter methylation. In conclusion, alterations in DNA methylation and differential expression of genes reveal that their potential functional interactions in TGEV infection. Our data highlights the epigenetic and transcriptomic landscapes in TGEV-infected ST cells and provides a reliable dataset for screening TGEV resistance genes and genetic markers.

Pollen Number and Ribosome Gene Expression Altered in a Genome-Editing Mutant of REDUCED POLLEN NUMBER1 Gene

The number of pollen grains varies within and between species. However, little is known about the molecular basis of this quantitative trait, in contrast with the many studies available on cell differentiation in the stamen. Recently, the first gene responsible for pollen number variation, REDUCED POLLEN NUMBER1 (RDP1), was isolated by genome-wide association studies of Arabidopsis thaliana and exhibited the signature of natural selection. This gene encodes a homolog of yeast Mrt4 (mRNA turnover4), which is an assembly factor of the large ribosomal subunit. However, no further data were available to link ribosome function to pollen development. Here, we characterized the RDP1 gene using the standard A. thaliana accession Col-0. The frameshift mutant, rdp1-3 generated by CRISPR/Cas9 revealed the pleiotropic effect of RDP1 in flowering, thus demonstrating that this gene is required for a broad range of processes other than pollen development. We found that the natural Col-0 allele conferred a reduced pollen number against the Bor-4 allele, as assessed using the quantitative complementation test, which is more sensitive than transgenic experiments. Together with a historical recombination event in Col-0, which was identified by sequence alignment, these results suggest that the coding sequence of RDP1 is the candidate region responsible for the natural phenotypic variation. To elucidate the biological processes in which RDP1 is involved, we conducted a transcriptome analysis. We found that genes responsible for ribosomal large subunit assembly/biogenesis were enriched among the differentially regulated genes, which supported the hypothesis that ribosome biogenesis is disturbed in the rdp1-3 mutant. Among the pollen-development genes, three key genes encoding basic helix-loop-helix (bHLH) transcription factors (ABORTED MICROSPORES (AMS), bHLH010, and bHLH089), as well as direct downstream genes of AMS, were downregulated in the rdp1-3 mutant. In summary, our results suggest a specialized function of ribosomes in pollen development through RDP1, which harbors natural variants under selection.

Mutations in Hcfc1 and Ronin result in an inborn error of cobalamin metabolism and ribosomopathy

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

Puf-A promotes cancer progression by interacting with nucleophosmin in nucleolus

Previously, we identified Puf-A as a novel member of Puf-family RNA-binding proteins; however, its biological functions remain obscure. Analysis of tumor samples of non-small cell lung cancer (NSCLC) showed that high Puf-A expression correlated with high histology grade and abnormal p53 status. Kaplan–Meier curve for overall survival revealed high expression of Puf-A to predict poor prognosis in stage I NSCLC. Among patients with colorectal cancer, high Puf-A expression also showed an adverse impact on overall survival. In lung cancer cell lines, downregulation of p53 increased Puf-A expression, and upregulation of p53 dampened its expression. However, luciferase reporter assays indicated that PUF-A locus harbored the p53-response element, but regulated Puf-A transcription indirectly. In vivo suppression of p53 in CCSP-rtTA/TetO-Cre/LSL-KrasG12D/p53flox/flox conditional mutant mice accelerated the progression of the KrasG12D-driven lung cancer, along with enhanced expression of Puf-A. Importantly, intranasal delivery of shPuf-A to the inducible KrasG12D/p53flox/flox mice suppressed tumor progression. Puf-A silencing led to marked decreases in the 80S ribosomes, along with decrease in S6 and L5 in the cytoplasm and accumulation in the nucleolus. Based on immunofluorescence staining and immunoprecipitation studies, Puf-A interacted with NPM1 in nucleolus. Puf-A silencing resulted in NPM1 translocation from nucleolus to nucleoplasm and this disruption of NPM1 localization was reversed by a rescue experiment. Mechanistically, Puf-A silencing altered NPM1 localization, leading to the retention of ribosomal proteins in nucleolus and diminished ribosome biogenesis, followed by cell-cycle arrest/cell death. Puf-A is a potential theranostic target for cancer therapy and an important player in cancer progression.

Screening of Bacillus velezensis E2 and the Inhibitory Effect of Its Antifungal Substances on Aspergillus flavus

Aspergilus flavus is the main pathogenic fungus that causes food mold. Effective control of A. flavus contamination is essential to ensure food safety. The lipopeptides (LPs) produced by Bacillus strains have been shown to have an obvious antifungal effect on molds. In this study, an antagonist strain of Bacillus velezensis with obvious antifungal activity against A. flavus was isolated from the surface of healthy rice. Using HPLC-MS analysis, the main components of LPs produced by strain E2 were identified as fengycin and iturins. Further investigations showed that LPs could inhibit the spore germination, and even cause abnormal expansion of hyphae and cell rupture. Transcriptomic analyses showed that some genes, involved in ribosome biogenesis in eukaryotes (NOG1, KRE33) and aflatoxin biosynthesis (aflK, aflR, veA, omtA) pathways in A. flavus were significantly down-regulated by LPs. In conclusion, this study provides novel insights into the cellular and molecular antifungal mechanisms of LPs against grain A. flavus contamination.

Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

Systematic analysis of the role of SLC52A2 in multiple human cancers

Background In humans, riboflavin must be obtained through intestinal absorption because it cannot be synthesized by the body. SLC52A2 encodes a membrane protein belonging to the riboflavin transporter protein family and is associated with a variety of diseases. Here, we systematically explore its relevance to multiple human tumors. Methods We analyzed the association of SLC52A2 with 33 tumors using publicly available databases such as TCGA and GEO. We verified the SLC52A2 expression in hepatocellular carcinoma, gastric cancer, colon cancer, and rectal cancer using immunohistochemistry. Results We report that SLC52A2 was highly expressed in almost all tumors, and the immunohistochemical results in the hepatocellular, gastric, colon, and rectal cancers were consistent with the above. SLC52A2 expression was linked to patient overall survival, disease-specific survival, progression-free interval, diagnosis, mutations, tumor mutational burden, microsatellite instability, common immune checkpoint genes, and immune cells infiltration. Enrichment analysis showed that SLC52A2 was mainly enriched in oocyte meiosis, eukaryotic ribosome biogenesis, and cell cycle. In hepatocellular carcinoma, the SLC52A2 expression is an independent prognostic factor. The SNHG3 and THUMPD3-AS1/hsa-miR-139-5p-SLC52A2 axis were identified as potential regulatory pathways in hepatocellular carcinoma. Conclusion In conclusion, we have systematically described for the first time that SLC52A2 is closely associated with a variety of tumors, especially hepatocellular carcinoma.

Viscoelastic RNA entanglement and advective flow underlie nucleolar form and function

The nucleolus facilitates transcription, processing, and assembly of ribosomal RNA (rRNA), the most abundant RNA in cells. Nucleolar function is facilitated by its multiphase liquid properties, but nucleolar fluidity and its connection to ribosome biogenesis remain unclear. Here, we used quantitative imaging, mathematical modeling, and pulse-chase nucleotide labelling to map nucleolar rRNA dynamics. Inconsistent with a purely diffusive process, rRNA steadily expands away from the transcriptional sites, moving in a slow (~1 Å/s), radially-directed fashion. This motion reflects the viscoelastic properties of a highly concentrated gel of entangled rRNA, whose constant polymerization drives steady outward flow. We propose a new viscoelastic rRNA release model, where nucleolar rRNA cleavage and processing reduce entanglement, fluidizing the nucleolar periphery to facilitate release of mature pre-ribosomal particles.

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.