Trace amine associated receptor 1: predicted effects of single nucleotide variants on structure-function in geographically diverse populations.

Trace Amine Associated Receptor 1 (TAAR1) is a novel pharmaceutical target under investigation for the treatment of several neuropsychiatric conditions. TAAR1 single nucleotide variants (SNV) have been found in patients with schizophrenia and metabolic disorders. However, the frequency of variants in geographically diverse populations and the functional effects of such variants are unknown. In this study, we aimed to characterise the distribution of TAAR1 SNVs in five different WHO regions using the Database of Genotypes and Phenotypes (dbGaP) and conducted a critical computational analysis using available TAAR1 structural data to identify SNVs affecting ligand binding and/or functional regions. Our analysis shows 19 orthosteric, 9 signalling and 16 micro-switch SNVs hypothesised to critically influence the agonist induced TAAR1 activation. These SNVs may non-proportionally influence populations from discrete regions and differentially influence the activity of TAAR1-targeting therapeutics in genetically and geographically diverse populations. Notably, our dataset presented with orthosteric SNVs D1033.32N (found only in the South-East Asian Region and Western Pacific Region) and T1945.42A (found only in South-East Asian Region), and 2 signalling SNVs (V1253.54A/T2526.36A, found in African Region and commonly, respectively), all of which have previously demonstrated to influence ligand induced functions of TAAR1. Furthermore, bioinformatics analysis using SIFT4G, MutationTaster 2, PROVEAN and MutationAssessor predicted all 16 micro-switch SNVs are damaging and may further influence the agonist activation of TAAR1, thereby possibly impacting upon clinical outcomes. Understanding the genetic basis of TAAR1 function and the impact of common mutations within clinical populations is important for the safe and effective utilisation of novel and existing pharmacotherapies.

Activation of Cryptic Donor Splice Sites within the UDP-Glucuronosyltransferase (UGT)1A First-Exon Region Generates Variant Transcripts That Encode UGT1A Proteins with Truncated Aglycone-Binding Domains.

The human UDP-glucuronosyltransferases (UGTs) have crucial roles in metabolizing and clearing numerous small lipophilic compounds. The UGT1A locus generates nine UGT1A mRNAs, 65 spliced transcripts, and 34 circular RNAs. In this study, our analysis of published UGT-RNA capture sequencing (CaptureSeq) datasets identified novel splice junctions that predict 24 variant UGT1A transcripts derived from ligation of exon 2 to unique sequences within the UGT1A first-exon region using cryptic donor splice sites. Of these variants, seven (1A1_n1, 1A3_n3, 1A4_n4, 1A5_n1, 1A8_n2, 1A9_n2, 1A10_n7) are predicted to encode UGT1A proteins with truncated aglycone-binding domains. We assessed their expression profiles and deregulation in cancer using four RNA sequencing (RNA-Seq) datasets of paired normal and cancerous drug-metabolizing tissues from large patient cohorts. Variants were generally coexpressed with their canonical counterparts with a higher relative abundance in tumor than in normal tissues. Variants showed tissue-specific expression with high interindividual variability but overall low abundance. However, 1A8_n2 showed high abundance in normal and cancerous colorectal tissues, with levels that approached or surpassed canonical 1A8 mRNA levels in many samples. We cloned 1A8_n2 and showed expression of the predicted protein (1A8_i3) in human embryonic kidney (HEK)293T cells. Glucuronidation assays with 4-methylumbelliferone (4MU) showed that 1A8_i3 had no activity and was unable to inhibit the activity of 1A8_i1 protein. In summary, the activation of cryptic donor splice sites within the UGT1A first-exon region expands the UGT1A transcriptome and proteome. The 1A8_n2 cryptic donor splice site is highly active in colorectal tissues, representing an important cis-regulatory element that negatively regulates the function of the UGT1A8 gene through pre-mRNA splicing. SIGNIFICANT STATEMENT: The UGT1A locus generates nine canonical mRNAs, 65 alternately spliced transcripts, and 34 different circular RNAs. The present study reports a series of novel UDP-glucuronosyltransferase (UGT)1A variants resulting from use of cryptic donor splice sites in both normal and cancerous tissues, several of which are predicted to encode variant UGT1A proteins with truncated aglycone-binding domains. Of these, 1A8_n2 shows exceptionally high abundance in colorectal tissues, highlighting its potential role in the first-pass metabolism in gut through the glucuronidation pathway.

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance.

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.

Deregulation of the Genes that Are Involved in Drug Absorption, Distribution, Metabolism, and Excretion in Hepatocellular Carcinoma.

Genes involved in drug absorption, distribution, metabolism, and excretion (ADME) are called ADME genes. Currently, 298 genes that encode phase I and II drug metabolizing enzymes, transporters, and modifiers are designated as ADME genes by the PharmaADME Consortium. ADME genes are highly expressed in the liver and their levels can be influenced by liver diseases such as hepatocellular carcinoma (HCC). In this study, we obtained RNA-sequencing and microRNA (miRNA)-sequencing data from 371 HCC patients via The Cancer Genome Atlas liver hepatocellular carcinoma project and performed ADME gene-targeted differential gene expression analysis and expression correlation analysis. Two hundred thirty-three of the 298 ADME genes (78%) were expressed in HCC. Of these genes, almost one-quarter (58 genes) were significantly downregulated, while only 6% (15) were upregulated in HCC relative to healthy liver. Moreover, one-half (14/28) of the core ADME genes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, NAT1, NAT2, UGT2B7, SLC22A1, SLCO1B1, and SLCO1B3) were downregulated. In addition, about one-half of the core ADME genes were positively correlated with each other and were also positively (AHR, ARNT, HNF4A, PXR, CAR, PPARA, and RXRA) or negatively (PPARD and PPARG) correlated with transcription factors known as ADME modifiers. Finally, we show that most miRNAs known to regulate core ADME genes are upregulated in HCC. Collectively, these data reveal 1) an extensive transcription factor-mediated ADME coexpression network in the liver that efficiently coordinates the metabolism and elimination of endogenous and exogenous compounds; and 2) a widespread deregulation of this network in HCC, most likely due to deregulation of both transcriptional and post-transcriptional (miRNA) pathways.

Identification of microRNA Biomarkers of Response to Neoadjuvant Chemoradiotherapy in Esophageal Adenocarcinoma Using Next Generation Sequencing.

BACKGROUND: Clinical trials report improved overall survival following neoadjuvant chemoradiotherapy in patients undergoing surgery for esophageal adenocarcinoma, with a 10-15% survival improvement. MicroRNAs (miRNAs) are small noncoding RNAs that are known to direct the behavior of cancers, including response to treatment. We investigated the ability of miRNAs to predict outcomes after neoadjuvant chemoradiotherapy. METHODS: Endoscopic biopsies from esophageal adenocarcinomas were obtained before neoadjuvant chemoradiotherapy and esophagectomy. miRNA levels were measured in the biopsies using next generation sequencing and compared with pathological response in the surgical resection, and subsequent survival. miRNA ratios that predicted pathological response were identified by Lasso regression and leave-one-out cross-validation. Association between miRNA ratio candidates and relapse-free survival was assessed using Kaplan-Meier analysis. Cox regression and Harrell's C analyses were performed to assess the predictive performance of the miRNAs. RESULTS: Two miRNA ratios (miR-4521/miR-340-5p and miR-101-3p/miR-451a) that predicted the pathological response to neoadjuvant chemoradiotherapy were found to be associated with relapse-free survival. Pretreatment expression of these two miRNA ratios, pretreatment tumor differentiation, posttreatment AJCC histopathological tumor regression grading, and posttreatment tumor clearance/margins were significant factors associated with survival in Cox regression analysis. Multivariate analysis of the two ratios together with pretherapy factors resulted in a risk prediction accuracy of 85% (Harrell's C), which was comparable with the prediction accuracy of the AJCC treatment response grading (77%). CONCLUSIONS: miRNA-ratio biomarkers identified using next generation sequencing can be used to predict disease free survival following neoadjuvant chemoradiotherapy and esophagectomy in patients with esophageal adenocarcinoma.

Barx2 and Pax7 Regulate Axin2 Expression in Myoblasts by Interaction with ß-Catenin and Chromatin Remodelling.

Satellite cells are the resident stem cells of skeletal muscle; quiescent in adults until activated by injury to generate proliferating myoblasts. The canonical Wnt signalling pathway, mediated by T-cell factor/lymphoid enhancer factor (TCF/LEF) and ß-catenin effector proteins, controls myoblast differentiation in vitro, and recent work suggests that timely termination of the Wnt/ß-catenin signal is important for normal adult myogenesis. We recently identified the Barx2 and Pax7 homeobox proteins as novel components of the Wnt effector complex. Here, we examine molecular and epigenetic mechanisms by which Barx2 and Pax7 regulate the canonical Wnt target gene Axin2, which mediates critical feedback to terminate the transcriptional response to Wnt signals. Barx2 is recruited to the Axin2 gene via TCF/LEF binding sites, recruits ß-catenin and the coactivator GRIP-1, and induces local H3K-acetylation. Barx2 also promotes nuclear localization of ß-catenin. Conversely, Pax7 represses Axin2 promoter/intron activity and inhibits Barx2-mediated H3K-acetylation via the corepressor HDAC1. Wnt3a not only induces Barx2 mRNA, but also stabilises Barx2 protein in myoblasts; conversely, Wnt3a potently inhibits Pax7 protein expression. As Barx2 promotes myogenic differentiation and Pax7 suppresses it, this novel posttranscriptional regulation of Barx2 and Pax7 by Wnt3a may be involved in the specification of differentiation-competent and -incompetent myoblast populations. Finally, we propose a model for dual function of Barx2 downstream of Wnt signals: activation of myogenic target genes in association with canonical myogenic regulatory factors, and regulation of the negative feedback loop that limits the response of myoblasts to Wnt signals via direct interaction of Barx2 with the TCF/ß-catenin complex. Stem Cells 2016;34:2169-2182.

A novel proteinase, SNOWY COTYLEDON4, is required for photosynthetic acclimation to higher light intensities in Arabidopsis.

Excess light can have a negative impact on photosynthesis; thus, plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the Arabidopsis (Arabidopsis thaliana) mutant snowy cotyledon4 (sco4) revealed a mutation in a chloroplast-targeted protein that shares limited homology with CaaX-type endopeptidases. The SCO4 protein possesses an important function in photosynthesis and development, with point mutations rendering the seedlings and adult plants susceptible to photooxidative stress. The sco4 mutation impairs the acclimation of chloroplasts and their photosystems to excess light, evidenced in a reduction in photosystem I function, decreased linear electron transfer, yet increased nonphotochemical quenching. SCO4 is localized to the chloroplasts, which suggests the existence of an unreported type of protein modification within this organelle. Phylogenetic and yeast complementation analyses of SCO4-like proteins reveal that SCO4 is a member of an unknown group of higher plant-specific proteinases quite distinct from the well-described CaaX-type endopeptidases RAS Converting Enzyme1 (RCE1) and zinc metallopeptidase STE24 and lacks canonical CaaX activity. Therefore, we hypothesize that SCO4 is a novel endopeptidase required for critical protein modifications within chloroplasts, influencing the function of proteins involved in photosynthesis required for tolerance to excess light.

Restricting Colorectal Cancer Cell Metabolism with Metformin: An Integrated Transcriptomics Study.

BACKGROUND: Metformin is a first-line therapy for type 2 diabetes as it disrupts cellular metabolism. Despite the association between metformin and lower cancer incidence, the anti-tumour activity of the drug in colorectal cancer (CRC) is incompletely understood. This study identifies underlying molecular mechanisms by which metformin slows colorectal cancer cell proliferation by investigating metformin-associated microRNA (miRNA) and target gene pairs implicated in signalling pathways. METHODS: The present study analysed changes in miRNAs and the coding transcriptome in CRC cells treated with a sublethal dose of metformin, followed by the contextual validation of potential miRNA-target gene pairs. RESULTS: Analyses of small RNA and transcriptome sequencing data revealed 104 miRNAs and 1221 mRNAs to be differentially expressed in CRC cells treated with metformin for 72 h. Interaction networks between differentially expressed miRNAs and putative target mRNAs were identified. Differentially expressed genes were mainly implicated in metabolism and signalling processes, such as the PI3K-Akt and MAPK/ERK pathways. Further validation of potential miRNA-target mRNA pairs revealed that metformin induced miR-2110 and miR-132-3p to target PIK3R3 and, consequently, regulate CRC cell proliferation, cell cycle progression and the PI3K-Akt signalling pathway. Metformin also induced miR-222-3p and miR-589-3p, which directly target STMN1 to inhibit CRC cell proliferation and cell cycle progression. CONCLUSIONS: This study identified novel changes in the coding transcriptome and small non-coding RNAs associated with metformin treatment of CRC cells. Integration of these datasets highlighted underlying mechanisms by which metformin impedes cell proliferation in CRC. Importantly, it identified the post-transcriptional regulation of specific genes that impact both metabolism and cell proliferation.

A Comprehensive Bioinformatic Analysis of RNA-seq Datasets Reveals a Differential and Variable Expression of Wildtype and Variant UGT1A Transcripts in Human Tissues and Their Deregulation in Cancers.

The UGT1A locus generates over 60 different alternatively spliced transcripts and 30 circular RNAs. To date, v2 and v3 transcripts are the only variant UGT1A transcripts that have been functionally characterized. Both v2 and v3 transcripts encode the same inactive variant UGT1A proteins (i2s) that can negatively regulate glucuronidation activity and influence cancer cell metabolism. However, the abundance and interindividual variability in the expression of v2 and v3 transcripts in human tissues and their potential deregulation in cancers have not been comprehensively assessed. To address this knowledge gap, we quantified the expression levels of v1, v2, and v3 transcripts using RNA-seq datasets with large cohorts of normal tissues and paired normal and tumor tissues from patients with six different cancer types (liver, kidney, colon, stomach, esophagus, and bladder cancer). We found that v2 and v3 abundance varied significantly between different tissue types, and that interindividual variation was also high within the same tissue type. Moreover, the ratio of v2 to v3 variants varied between tissues, implying their differential regulation. Our results showed higher v2 abundance in gastrointestinal tissues than liver and kidney tissues, suggesting a more significant negative regulation of glucuronidation by i2 proteins in gastrointestinal tissues than in liver and kidney tissues. We further showed differential deregulation of wildtype (v1) and variant transcripts (v2, v3) in cancers that generally increased the v2/v1 and/or v3/v1 expression ratios in tumors compared to normal tissues, indicating a more significant role of the variants in tumors. Finally, we report ten novel UGT1A transcripts with novel 3' terminal exons, most of which encode variant proteins with a similar structure to UGT1A_i2 proteins. These findings further emphasize the diversity of the UGT1A transcriptome and proteome.

SARS-CoV-2 produces a microRNA CoV2-miR-O8 in patients with COVID-19 infection.

Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs.

Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs.

Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5' and 3' ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

The Somatic Mutation Landscape of UDP-Glycosyltransferase (UGT) Genes in Human Cancers.

The human UDP-glycosyltransferase (UGTs) superfamily has a critical role in the metabolism of anticancer drugs and numerous pro/anti-cancer molecules (e.g., steroids, lipids, fatty acids, bile acids and carcinogens). Recent studies have shown wide and abundant expression of UGT genes in human cancers. However, the extent to which UGT genes acquire somatic mutations within tumors remains to be systematically investigated. In the present study, our comprehensive analysis of the somatic mutation profiles of 10,069 tumors from 33 different TCGA cancer types identified 3427 somatic mutations in UGT genes. Overall, nearly 18% (1802/10,069) of the assessed tumors had mutations in UGT genes with huge variations in mutation frequency across different cancer types, ranging from over 25% in five cancers (COAD, LUAD, LUSC, SKCM and UCSC) to less than 5% in eight cancers (LAML, MESO, PCPG, PAAD, PRAD, TGCT, THYM and UVM). All 22 UGT genes showed somatic mutations in tumors, with UGT2B4, UGT3A1 and UGT3A2 showing the largest number of mutations (289, 307 and 255 mutations, respectively). Nearly 65% (2260/3427) of the mutations were missense, frame-shift and nonsense mutations that have been predicted to code for variant UGT proteins. Furthermore, about 10% (362/3427) of the mutations occurred in non-coding regions (5' UTR, 3' UTR and splice sites) that may be able to alter the efficiency of translation initiation, miRNA regulation or the splicing of UGT transcripts. In conclusion, our data show widespread somatic mutations of UGT genes in human cancers that may affect the capacity of cancer cells to metabolize anticancer drugs and endobiotics that control pro/anti-cancer signaling pathways. This highlights their potential utility as biomarkers for predicting therapeutic efficacy and clinical outcomes.

Integrative Transcriptomic Network Analysis of Butyrate Treated Colorectal Cancer Cells.

Diet-derived histone deacetylase inhibitor (HDACi), butyrate, alters global acetylation and consequently global gene expression in colorectal cancer (CRC) cells to exert its anticancer effects. Aberrant microRNA (miRNA) expression contributes to CRC development and progression. Butyrate-mediated modulation of microRNA (miRNA) expression remains under-investigated. This study employed a systems biology approach to gain a comprehensive understanding of the complex miRNA-mRNA interactions contributing to the butyrate response in CRC cells. Next-generation sequencing, gene ontology (GO) and pathway enrichment analyses were utilized to reveal the extent of butyrate-mediated gene regulation in CRC cells. Changes in cell proliferation, apoptosis, the cell cycle and gene expression induced by miRNAs and target gene knockdown in CRC cells were assessed. Butyrate induced differential expression of 113 miRNAs and 2447 protein-coding genes in HCT116 cells. Butyrate also altered transcript splicing of 1591 protein-coding genes. GO, and pathway enrichment analyses revealed the cell cycle to be a central target of the butyrate response. Two butyrate-induced miRNAs, miR-139 and miR-542, acted cooperatively with butyrate to induce apoptosis and reduce CRC cell proliferation by regulating target genes, including cell cycle-related EIF4G2 and BIRC5. EIF4G2 RNA interference mimicked the miR-139-mediated reduction in cell proliferation. The cell cycle is a critical pathway involved in the butyrate response of CRC cells. These findings reveal novel roles for miRNAs in the cell cycle-related, anticancer effects of butyrate in CRC cells.

The Expression Profiles and Deregulation of UDP-Glycosyltransferase (UGT) Genes in Human Cancers and Their Association with Clinical Outcomes.

The human UDP-glycosyltransferase (UGTs) superfamily has 22 functional enzymes that play a critical role in the metabolism of small lipophilic compounds, including carcinogens, drugs, steroids, lipids, fatty acids, and bile acids. The expression profiles of UGT genes in human cancers and their impact on cancer patient survival remains to be systematically investigated. In the present study, a comprehensive analysis of the RNAseq and clinical datasets of 9514 patients from 33 different TCGA (the Genome Cancer Atlas) cancers demonstrated cancer-specific UGT expression profiles with high interindividual variability among and within individual cancers. Notably, cancers derived from drug metabolizing tissues (liver, kidney, gut, pancreas) expressed the largest number of UGT genes (COAD, KIRC, KIRP, LIHC, PAAD); six UGT genes (1A6, 1A9, 1A10, 2A3, 2B7, UGT8) showed high expression in five or more different cancers. Kaplan-Meier plots and logrank tests revealed that six UGT genes were significantly associated with increased overall survival (OS) rates [UGT1A1 (LUSC), UGT1A6 (ACC), UGT1A7 (ACC), UGT2A3 (KIRC), UGT2B15 (BLCA, SKCM)] or decreased OS rates [UGT2B15 (LGG), UGT8 (UVM)] in specific cancers. Finally, differential expression analysis of 611 patients from 12 TCGA cancers identified 16 UGT genes (1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2A3, 2B4, 2B7, 2B11, 2B15, 3A1, 3A2, UGT8) that were up/downregulated in at least one cancer relative to normal tissues. In conclusion, our data show widespread expression of UGT genes in cancers, highlighting the capacity for intratumoural drug metabolism through the UGT conjugation pathway. The data also suggests the potentials for specific UGT genes to serve as prognostic biomarkers or therapeutic targets in cancers.

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development.

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a ζ-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1, thereby controlling plastid development.

SRRM4 Expands the Repertoire of Circular RNAs by Regulating Microexon Inclusion.

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)-which occur in 30% of functional mRNA transcripts-have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs. In addition, when employing a bespoke bioinformatics pipeline for identifying RNA chimeras, called Hyb, we could also identify over 2000 ME-circRNAs which contain novel MEs at their backsplice junctions, that are uncalled by either CIRCexplorer2 or CIRI2. Analysis of circRNA-seq datasets from gliomas of varying clinical grades compared with matched control tissue has shown circRNAs have potential as prognostic markers for stratifying tumor from healthy tissue. Furthermore, the abundance of microexon-containing circRNAs (ME-circRNAs) between tumor and normal tissues is correlated with the expression of a splicing associated factor, Serine/arginine repetitive matrix 4 (SRRM4). Overexpressing SRRM4, known for regulating ME inclusion in mRNAs critical for neural differentiation, in human HEK293 cells resulted in the biogenesis of over 2000 novel ME-circRNAs, including ME-circEIF4G3, and changes in the abundance of many canonical circRNAs, including circSETDB2 and circLBRA. This shows SRRM4, in which its expression is correlated with poor prognosis in gliomas, acts as a bona fide circRNA biogenesis factor. Given the known roles of MEs and circRNAs in oncogenesis, the identification of these previously unrecognized ME-circRNAs further increases the complexity and functional purview of this non-coding RNA family.

Expression of microRNA in human retinal pigment epithelial cells following infection with Zaire ebolavirus.

OBJECTIVE: Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation-or uveitis-which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection. RESULTS: Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors.

Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978.

In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4',6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeB and ΔadeAB cells augmented results seen for ΔadeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRS revealed transcription of 290 genes were ≥2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.

Southern South Australian groundwater microbe diversity.

Groundwater is increasingly used globally for domestic, industrial and agricultural production. While many studies have focused on groundwater as a resource, the diverse ecosystems within are often ignored. Here, we assess 54 Southern South Australian groundwater microbial communities from the populated part of the state to assess their status and dynamics in isolated groundwater systems. We observed a strong site-to-site individuality in groundwater bacterial communities, likely due to the isolated nature of groundwater bodies leading to unique ecosystems. Rank abundance analysis indicates bacterial diversity is maintained even at low abundances and that the distribution fits classical ecological models for strong competition in resource-limited environments. Combined, our data indicates that despite overrepresentation of pollutant-associated bacterial orders in and around the Adelaide metropolitan area, microbial communities remain diverse and show little evidence of converging on a common pollutant-effected community.