Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms

m6A methylation modulates adipogenesis through JAK2-STAT3-C/EBPβ signaling Publication date: August 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 8 Author(s): Ruifan Wu, Guanqun Guo, Zhen Bi, Youhua Liu, Yuanling Zhao, Nana Chen, Fengqin Wang, Yizhen Wang, Xinxia Wang Abstract N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Here, we reveal that deletion of m6A demethylase FTO in porcine and mouse preadipocytes inhibits adipogenesis through JAK2-STAT3-C/EBPβ signaling. Mechanistically, FTO deficiency suppresses JAK2 expression and STAT3 phosphorylation, leading to attenuated transcription of C/EBPβ, which is essential for the early stage of adipocyte differentiation. Using dual-luciferase assay, we validate that knockdown of FTO reduces expression of JAK2 in an m6A-dependent manner. Furthermore, we find that m6A “reader” protein YTHDF2 directly targets m6A-modified transcripts of JAK2 and accelerates mRNA decay, which results in decreased JAK2 expression and inactivated JAK2-STAT3-C/EBPβ signaling, thereby inhibiting adipogenesis. Collectively, our results provide a novel insight into the molecular mechanism of m6A methylation in post-transcriptional regulation of JAK2-STAT3-C/EBPβ signaling axis and highlight the crucial role of m6A modification and its modulators in adipogenesis.

Hypoxia-induced RelA/p65 derepresses SLC16A3 (MCT4) by downregulating ZBTB7A Publication date: August 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 8 Author(s): Seo-Hyun Choi, Min-Young Kim, Young-So Yoon, Dong-In Koh, Min-Kyeong Kim, Su-Yeon Cho, Kyung-Sup Kim, Man-Wook Hur Abstract Overexpressed Solute Carrier Family 16 Member 3 (SLC16A3, also called MCT4) plays a critical role in hypoxic cancer cell growth and proliferation, by expelling glycolysis-derived lactate across the plasma membrane. However, how SLC16A3 expression is regulated, under hypoxic conditions, is poorly understood. FBI-1, encoded by ZBTB7A, is a proto-oncoprotein. Interestingly, under hypoxic conditions, expression of SLC16A3, and hypoxia-inducible factor-1 (HIF-1), increased gradually, while FBI-1 expression decreased, suggesting a negative correlation between SLC16A3/HIF-1 and FBI-1 expression. Consequently, we hypothesized that FBI-1 might regulate SLC16A3 and/or HIF-1 expression. Transient transfection and transcription assays of SLC16A3 promoter reporter fusion constructs, oligonucleotide-pulldowns, and ChIP assays, showed that HIF-1α activates SLC16A3 by binding to a hypoxia-response element (HRE), while ectopic FBI-1 potently repressed SLC16A3, by binding to both FBI-1-response elements (FREs) and HREs, during hypoxia. Further evidence for this model was downregulation of ZBTB7A, correlated with SLC16A3 upregulation, in hypoxic colon cancer cells. We also investigated how FBI-1 expression is downregulated during hypoxia. The 5′-upstream regulatory region of ZBTB7A contains two NF-κB-binding sites and two HREs. Interestingly, hypoxia activated NF-κB (RelA/p65) and also increased its nuclear translocation. NF-κB repressed ZBTB7A by binding NF-κB-binding elements, and downregulated the repressor FBI-1, thereby increasing SLC16A3 transcription. While transcriptional repression of SLC16A3 by FBI-1 inhibited lactate efflux, repression of ZBTB7A and activation of lactate efflux by NF-κB, increased colon cancer cell growth and proliferation.

KSRP modulates melanoma growth and efficacy of vemurafenib Publication date: August 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 8 Author(s): Wenwen Liu, Chu-Fang Chou, Shanrun Liu, David Crossman, Nabiha Yusuf, Yunkun Wu, Ching-Yi Chen Abstract The majority of melanomas carry an oncogenic BRAF mutation (BRAFV600E), which results in constitutive kinase activity driving melanoma proliferation. While inhibitors of BRAFV600E (BRAFi) effectively lead to rapid tumor shrinkage, most patients treated with BRAFi develop acquired resistance. Identification of factors as regulators of melanoma growth and as potential sources of resistance is thus crucial for the design of improved therapies to treat advanced melanoma with more durable responses. Here, we show that KH-type splicing regulatory protein (KSRP) is critical for proliferation of melanoma cells without and with acquired resistance to vemurafenib. Silencing KSRP reduces cell proliferation and augments the growth suppressive effects of vemurafenib. We identify killin (KLLN), a p53-regulated DNA replication inhibitor, as a downstream effector of growth inhibition by KSRP silencing and demonstrate that KSRP promotes decay of KLLN mRNA through an RNA-protein interaction. Using heterologous mRNA reporters, we show that a U-rich element within the 3′ untranslated region of KLLN is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of melanoma cell growth in part through controlling KLLN mRNA stability.

Temperature-dependent regulation of the Escherichia coli lpxT gene Publication date: August 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 8 Author(s): Barbara Sciandrone, Francesca Forti, Sara Perego, Federica Falchi, Federica Briani Abstract The Lipid A moiety of the lipopolysaccharide can be covalently modified during its transport to the outer membrane by different enzymes, among which the LpxT inner membrane protein. LpxT transfers a phosphate group from the undecaprenyl pyrophosphate to the Lipid A, a modification affecting the stability of the outer membrane and its recognition by the host immune system in Enterobacteria. We previously found that the expression of the Pseudomonas aeruginosa lpxT gene, encoding LpxT, is induced in response to a temperature upshift and we proposed that an RNA thermometer was responsible for such regulation. Here we show that the Escherichia coli lpxT orthologous gene is down-regulated upon a temperature upshift and investigated the mechanism of this regulation. We found that the LpxT protein stability is not affected by the temperature change. Conversely, the lpxT mRNA levels strongly decrease upon a shift from 28 to 42 °C. The lack of MicA sRNA, which was previously implicated in lpxT regulation, does not affect lpxT thermal regulation. We identified the lpxTp promoter and demonstrated that lpxTp has temperature-sensitive activity depending on its peculiar −10 region. Moreover, we found that RNase E-dependent degradation of the lpxT mRNA is also modulated by temperature causing a strong destabilization of the lpxT mRNA at 42 °C. In vitrodata argue against the involvement of factors differentially expressed at 28 and 42 °C in the temperature–dependent modulation of lpxT mRNA stability.

Small changes, big implications: The impact of m6A RNA methylation on gene expression in pluripotency and development Publication date: Available online 17 July 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Adam M. Heck, Carol J. Wilusz Abstract In order to maintain a state of self-renewal, yet retain the ability to rapidly differentiate in response to external signals, pluripotent cells exert tight control over gene expression at many levels. Recent studies have suggested that N6-methyladenosine (m6A) RNA methylation, one of the most abundant post-transcriptional modifications, is important for both pluripotency and differentiation. In this review, we summarize the current state of the m6A field, with emphasis on the impact of writers, erasers and readers of m6A on RNA metabolism and stem cell biology.

Small non-coding RNA within the endogenous spliceosome and alternative splicing regulation Publication date: Available online 16 July 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Ruth Sperling Abstract Splicing and alternative splicing (AS), which occur in the endogenous spliceosome, play major roles in regulating gene expression, and defects in them are involved in numerous human diseases including cancer. Although the mechanism of the splicing reaction is well understood, the regulation of AS remains to be elucidated. A group of essential regulatory factors in gene expression are small non-coding RNAs (sncRNA): e.g. microRNA, mainly known for their inhibitory role in translation in the cytoplasm; and small nucleolar RNA, known for their role in methylating non-coding RNA in the nucleolus. Here I highlight a new aspect of sncRNAs found within the endogenous spliceosome. Assembled in non-canonical complexes and through different base pairing than their canonical ones, spliceosomal sncRNAs can potentially target different RNAs. Examples of spliceosomal sncRNAs regulating AS, regulating gene expression, and acting in a quality control of AS are reviewed, suggesting novel functions for spliceosomal sncRNAs. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

Repeat-associated RNA structure and aberrant splicing Publication date: Available online 16 July 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Melissa A. Hale, Nicholas E. Johnson, J. Andrew Berglund Abstract Over 30 hereditary disorders attributed to the expansion of microsatellite repeats have been identified. Despite variant nucleotide content, number of consecutive repeats, and different locations in the genome, many of these diseases have pathogenic RNA gain-of-function mechanisms. The repeat-containing RNAs can form structures in vitro predicted to contribute to the disease through assembly of intracellular RNA aggregates termed foci. The expanded repeat RNAs within these foci sequester RNA binding proteins (RBPs) with important roles in the regulation of RNA metabolism, most notably alternative splicing (AS). These deleterious interactions lead to downstream alterations in transcriptome-wide AS directly linked with disease symptoms. This review summarizes existing knowledge about the association between the repeat RNA structures and RBPs as well as the resulting aberrant AS patterns, specifically in the context of myotonic dystrophy. The connection between toxic, structured RNAs and dysregulation of AS in other repeat expansion diseases is also discussed. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

HOXA2 activity regulation by cytoplasmic relocation, protein stabilization and post-translational modification Publication date: Available online 16 July 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Noémie Deneyer, Laure Bridoux, Céline Bombled, Tamara Pringels, Isabelle Bergiers, Sébastien Pyr dit Ruys, Didier Vertommen, Jean-Claude Twizere, René Rezsohazy Abstract HOX proteins are homeodomain transcription factors critically involved in patterning animal embryos and controlling organogenesis. While the functions of HOX proteins and the processes under their control begin to be well documented, the modalities of HOX protein activity regulation remain poorly understood. Here we show that HOXA2 interacts with PPP1CB, a catalytic subunit of the Ser/Thr PP1 phosphatase complex. This interaction co-localizes in the cytoplasm with a previously described HOXA2 interactor, KPC2, which belongs to the KPC E3 ubiquitin ligase complex. We provide evidence that HOXA2, PPP1CB and KPC2 define a molecularly and functionally interacting complex. Collectively, our experiments support that PPP1CB and KPC2 together inhibit the activity of HOXA2 by activating its nuclear export, but favored HOXA2 de-ubiquitination and stabilization thereby establishing a store of HOXA2 in the cytoplasm.

How RNA structure dictates the usage of a critical exon of spinal muscular atrophy gene Publication date: Available online 16 July 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Natalia N. Singh, Ravindra N. Singh Abstract Role of RNA structure in pre-mRNA splicing has been implicated for several critical exons associated with genetic disorders. However, much of the structural studies linked to pre-mRNA splicing regulation are limited to terminal stem-loop structures (hairpins) sequestering splice sites. In few instances, role of long-distance interactions is implicated as the major determinant of splicing regulation. With the recent surge of reports of circular RNA (circRNAs) generated by backsplicing, role of Alu-associated RNA structures formed by long-range interactions are taking central stage. Humans contain two nearly identical copies of Survival Motor Neuron (SMN) genes, SMN1 and SMN2. Deletion or mutation of SMN1 coupled with inability of SMN2 to compensate for the loss of SMN1 due to exon 7 causes spinal muscular atrophy (SMA), one of the leading genetic diseases of children. In this review, we describe how structural elements formed by both local and long-distance interactions are being exploited to modulate SMN2 exon 7 splicing as a potential therapy for SMA. We also discuss how Alu-associated secondary structure modulate generation of a vast repertoire of SMN circRNAs. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

The regulation properties of RNA secondary structure in alternative splicing Publication date: Available online 16 July 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Natalia Bartys, Ryszard Kierzek, Jolanta Lisowiec-Wachnicka Abstract The RNA secondary structure is important for many functional processes in the cell. The secondary and tertiary structures of cellular RNAs are essential for the activity of these molecules in processes such as transcription, splicing, translation, and localization. New high-throughput analytical methods, including next generation sequencing, have allowed for the in-depth characterization of the ‘RNA structurome’: a new term describing how the RNA structure controls the activity of RNA by itself and how it regulates the expression of genes. In this review, we present many examples of the influence of structural motifs of RNA, long range interactions and global RNA structure on the alternative splicing processes. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.