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Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.
Triple negative breast cancers (TNBC) are aggressive tumors, with high rates of metastatic spread and targeted therapies are critically needed. We aimed to assess the prognostic and predictive value of a 3q 19-gene signature identified previously from lung cancer in a collection of 4,801 breast tumor gene expression data. The 3q gene signature had a strong association with features of aggressiveness such as high grade, hormone receptor negativity, presence of a basal-like or TNBC phenotype and reduced distant metastasis free survival. The 3q gene signature was strongly associated with lung metastasis only in TNBC (P < 0.0001, Hazard ratio (HR) 1.44, 95% confidence interval (CI), 1.31-1.60), significantly associated with brain but not bone metastasis regardless of TNBC status. The association of one 3q driver gene FXR1 with distant metastasis in TNBC (P = 0.01) was further validated by immunohistochemistry. In addition, the 3q gene signature was associated with better response to neoadjuvant chemotherapy in TNBC (P < 0.0001) but not in non-TNBC patients. Our study suggests that the 3q gene signature is a novel prognostic marker for lung and/or brain metastasis and a predictive marker for the response to neoadjuvant chemotherapy in TNBC, implying a potential role for 3q genes in the mechanism of organ-specific metastasis.
Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer's vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function.
© 2017 Jao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
The importance of epistasis-or statistical interactions between genetic variants-to the development of complex disease in humans has been controversial. Genome-wide association studies of statistical interactions influencing human traits have recently become computationally feasible and have identified many putative interactions. However, statistical models used to detect interactions can be confounded, which makes it difficult to be certain that observed statistical interactions are evidence for true molecular epistasis. In this study, we investigate whether there is evidence for epistatic interactions between genetic variants within the cis-regulatory region that influence gene expression after accounting for technical, statistical, and biological confounding factors. We identified 1,119 (FDR = 5%) interactions that appear to regulate gene expression in human lymphoblastoid cell lines, a tightly controlled, largely genetically determined phenotype. Many of these interactions replicated in an independent dataset (90 of 803 tested, Bonferroni threshold). We then performed an exhaustive analysis of both known and novel confounders, including ceiling/floor effects, missing genotype combinations, haplotype effects, single variants tagged through linkage disequilibrium, and population stratification. Every interaction could be explained by at least one of these confounders, and replication in independent datasets did not protect against some confounders. Assuming that the confounding factors provide a more parsimonious explanation for each interaction, we find it unlikely that cis-regulatory interactions contribute strongly to human gene expression, which calls into question the relevance of cis-regulatory interactions for other human phenotypes. We additionally propose several best practices for epistasis testing to protect future studies from confounding.
Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Repeated challenge of lipopolysaccharide (LPS) alters the response to subsequent LPS exposures via modulation of toll-like receptor 4 (TLR4). Whether activation of other TLRs can modulate TLR4 responses, and vice versa, remains unclear. Specifically with regards to endothelial cells, a key component of innate immunity, the impact of TLR cross-modulation is unknown. We postulated that TLR2 priming (via Pam3Csk4) would inhibit TLR4-mediated responses while TLR3 priming (via Poly I:C) would enhance subsequent TLR4-inflammatory signaling. We studied human umbilical vein endothelial cells (HUVECs) and neonatal human dermal microvascular endothelial cells (HMVECs). Cells were primed with a combination of Poly I:C (10 μg/ml), Pam3Csk4 (10 μg/ml), or LPS (100 ng/ml), then washed and allowed to rest. They were then rechallenged with either Poly I:C, Pam3Csk4 or LPS. Endothelial cells showed significant tolerance to repeated LPS challenge. Priming with Pam3Csk4 also reduced the response to secondary LPS challenge in both cell types, despite a reduced proinflammatory response to Pam3Csk4 in HMVECs compared to HUVECs. Poly I:C priming enhanced inflammatory and interferon producing signals upon Poly I:C or LPS rechallenge, respectively. Poly I:C priming induced interferon regulatory factor 7, leading to enhancement of interferon production. Finally, both Poly I:C and LPS priming induced significant changes in receptor-interacting serine/threonine-protein kinase 1 activity. Pharmacological inhibition of receptor-interacting serine/threonine-protein kinase 1 or interferon regulatory factor 7 reduced the potentiated phenotype of TLR3 priming on TLR4 rechallenge. These results demonstrate that in human endothelial cells, prior activation of TLRs can have a significant impact on subsequent exposures and may contribute to the severity of the host response.
Copyright © 2016 Elsevier Inc. All rights reserved.
RNA-binding protein (RBP) is a key player in regulating gene expression at the posttranscriptional level. CLIP-Seq, with the ability to provide a genome-wide map of protein-RNA interactions, has been increasingly used to decipher RBP-mediated posttranscriptional regulation. Generating highly reliable binding sites from CLIP-Seq requires not only stringent library preparation but also considerable computational efforts. Here we presented a first systematic evaluation of major computational steps for identifying RBP binding sites from CLIP-Seq data, including preprocessing, the choice of control samples, peak normalization, and motif discovery. We found that avoiding PCR amplification artifacts, normalizing to input RNA or mRNAseq, and defining the background model from control samples can reduce the bias introduced by RNA abundance and improve the quality of detected binding sites. Our findings can serve as a general guideline for CLIP experiments design and the comprehensive analysis of CLIP-Seq data.
Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.
Published by Elsevier Ltd.
Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3'UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.
CD44E is a frequently overexpressed variant of CD44 in gastric cancer. Mechanisms that regulate CD44 splicing and expression in gastric cancer remain unknown. Herein, we investigated the role of DARPP-32 (dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000) in promoting tumor growth through regulation of CD44 splicing. Using western blot and quantitative real-time PCR analysis, our results indicated that knockdown of endogenous DARPP-32 markedly reduces the expression of CD44 V8-V10 (CD44E). Using a quantitative splicing luciferase reporter system, we detected a significant increase in the reporter activity following DARPP-32 overexpression (P<0.001). Conversely, knocking down endogenous DARPP-32 significantly attenuated the splicing activity (P<0.001). Further experiments showed that DARPP-32 regulates the expression of SRp20 splicing factor and co-exists with it in the same protein complex. Inhibition of alternative splicing with digitoxin followed by immunoprecipitation and immunoblotting indicated that DARPP-32 has an important role in regulating SRp20 protein stability. The knockdown of endogenous DARPP-32 confirmed that DARPP-32 regulates the SRp20-dependent CD44E splicing. Using tumor xenograft mouse model, knocking down endogenous DARPP-32 markedly reduced SRp20 and CD44E protein levels with a decreased tumor growth. The reconstitution of SRp20 expression in these cells rescued tumor growth. In addition, we also demonstrated frequent co-overexpression and positive correlation of DARPP-32, SRp20 and CD44E expression levels in human gastric primary tumors. Our novel findings establish for the first time the role of DARPP-32 in regulating splicing factors in gastric cancer cells. The DARPP-32-SRp20 axis has a key role in regulating the CD44E splice variant that promotes gastric tumorigenesis.
Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fenton's reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components caspase-1/11 or Nlrp3 or by inhibition of caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.