Y14 governs p53 expression and modulates DNA damage sensitivity.
Lu Chia-Chen,Lee Chi-Chieh,Tseng Ching-Tzu,Tarn Woan-Yuh
Y14 is a core component of the exon junction complex (EJC), while it also exerts cellular functions independent of the EJC. Depletion of Y14 causes G2/M arrest, DNA damage and apoptosis. Here we show that knockdown of Y14 induces the expression of an alternative spliced isoform of p53, namely p53β, in human cells. Y14, in the context of the EJC, inhibited aberrant exon inclusion during the splicing of p53 pre-mRNA, and thus prevent p53β expression. The anti-cancer agent camptothecin specifically suppressed p53β induction. Intriguingly, both depletion and overexpression of Y14 increased overall p53 protein levels, suggesting that Y14 governs the quality and quantity control of p53. Moreover, Y14 depletion unexpectedly reduced p21 protein levels, which in conjunction with aberrant p53 expression accordingly increased cell sensitivity to genotoxic agents. This study establishes a direct link between Y14 and p53 expression and suggests a function for Y14 in DNA damage signaling.
The genes encoding the type II gonadotropin-releasing hormone receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci.
Faurholm B,Millar R P,Katz A A
We have cloned and characterized two genomic loci encoding the human type II gonadotropin-releasing hormone (GnRH) receptor and RNA-binding motif protein-8 (RBM8A). In both loci the genes overlap and are in antisense orientation to each other. The locus on chromosome 1 encompasses the type II GnRH receptor gene (GNRHR2), which is composed of three exons. We found transcripts from this gene in a wide range of tissues, but they lacked a methionine initiation codon and had a stop codon in exon 2. In the antisense orientation, this locus contains RBM8A, which consists of six exons and directs the synthesis of an RBM8A protein of 173 or 174 amino acids by alternative splicing. A second locus on chromosome 14 contains pseudogenes of RBM8A and the type II GnRH receptor and probably originated from the chromosome 1 locus by retrotransposition.
Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model.
Sudo Hitomi,Tsuji Atsushi B,Sugyo Aya,Kohda Masakazu,Sogawa Chizuru,Yoshida Chisato,Harada Yoshi-nobu,Hino Okio,Saga Tsuneo
Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes-COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A-would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.
Depletion of RNA-binding protein RBM8A (Y14) causes cell cycle deficiency and apoptosis in human cells.
Ishigaki Yasuhito,Nakamura Yuka,Tatsuno Takanori,Hashimoto Mitsumasa,Shimasaki Takeo,Iwabuchi Kuniyoshi,Tomosugi Naohisa
Experimental biology and medicine (Maywood, N.J.)
RBM8A (Y14) contains an RNA-binding motif and forms a tight heterodimer with Magoh. The heterodimer is known to be a member of the exon junction complex that forms on mRNA before export and it is required for mRNA metabolism processes such as splicing, mRNA export and nonsense-mediated mRNA decay. Recently, deficient cellular proliferation has been observed in RBM8A- or Magoh-depleted cells. These results prompted us to study the role of RBM8A in cell cycle progression of human tumour cells. The depletion of RBM8A in A549 cells resulted in poor cell survival and the accumulation of mitotic cells. After release from G1/S arrest induced by a double thymidine block, the RBM8A-silenced cells could not proceed to the next G1 phase beyond G2/M phase. Finally, the sub-G1 population increased and the apoptosis markers caspases 3/7 were activated. Silenced cells exhibited an increased frequency of multipolar or monopolar centrosomes, which may have caused the observed deficiency in cell cycle progression. Finally, silencing of either RBM8A or Magoh resulted in mutual downregulation of the other protein. These results illustrate that the RBM8A-Magoh mRNA binding complex is required for M phase progression and both proteins may be novel targets for anticancer therapy.
Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh.
Ishigaki Yasuhito,Nakamura Yuka,Tatsuno Takanori,Ma Shaofu,Tomosugi Naohisa
Experimental biology and medicine (Maywood, N.J.)
The RNA-binding protein 8A (RBM8A)-mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect in vitro and in vivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.
Rbm8a haploinsufficiency disrupts embryonic cortical development resulting in microcephaly.
Mao Hanqian,Pilaz Louis-Jan,McMahon John J,Golzio Christelle,Wu Danwei,Shi Lei,Katsanis Nicholas,Silver Debra L
The Journal of neuroscience : the official journal of the Society for Neuroscience
The cerebral cortex is built during embryonic neurogenesis, a period when excitatory neurons are generated from progenitors. Defects in neurogenesis can cause acute neurodevelopmental disorders, such as microcephaly (reduced brain size). Altered dosage of the 1q21.1 locus has been implicated in the etiology of neurodevelopmental phenotypes; however, the role of 1q21.1 genes in neurogenesis has remained elusive. Here, we show that haploinsufficiency for Rbm8a, an exon junction complex (EJC) component within 1q21.1, causes severe microcephaly and defective neurogenesis in the mouse. At the onset of neurogenesis, Rbm8a regulates radial glia proliferation and prevents premature neuronal differentiation. Reduced Rbm8a levels result in subsequent apoptosis of neurons, and to a lesser extent, radial glia. Hence, compared to control, Rbm8a-haploinsufficient brains have fewer progenitors and neurons, resulting in defective cortical lamination. To determine whether reciprocal dosage change of Rbm8a alters embryonic neurogenesis, we overexpressed human RBM8A in two animal models. Using in utero electroporation of mouse neocortices as well as zebrafish models, we find RBM8A overexpression does not significantly perturb progenitor number or head size. Our findings demonstrate that Rbm8a is an essential neurogenesis regulator, and add to a growing literature highlighting roles for EJC components in cortical development and neurodevelopmental pathology. Our results indicate that disruption of RBM8A may contribute to neurodevelopmental phenotypes associated with proximal 1q21.1 microdeletions.
High expression of RBM8A predicts poor patient prognosis and promotes tumor progression in hepatocellular carcinoma.
Liang Rong,Lin Yan,Ye Jia-Zhou,Yan Xue-Xin,Liu Zhi-Hui,Li Yong-Qiang,Luo Xiao-Ling,Ye Hai-Hong
Hepatocellular carcinoma (HCC) is a huge threat for human health worldwide. As a complicated tumor, the molecular basis for HCC development especially metastasis requires exploration. Although RNA binding motif (RBM) proteins are closely related to various cancers, the clinical importance and underlying mechanisms of RBM8A in HCC remain elusive. In this study, we found that RBM8A was highly expressed in HCC tumor tissues compared to normal liver tissues. Overexpression of RBM8A was associated with HbsAg and Edmondson pathological grading. Moreover, Kaplan-Meier survival analysis showed that high expression of RBM8A was related to the poor overall survival and progression-free survival of patients with HCC. Gain- and loss-of-function experiments further demonstrated that RBM8A promoted tumor cell migration and invasion in HCC via activation of epithelial-mesenchymal transition signaling pathway. It is also noteworthy that RBM8A is required for tumor cell proliferation and anti-apoptosis in HCC. Altogether, our results revealed a close relationship between RBM8A and HCC prognosis as well as a critical tumor-promoting function of RBM8A in HCC progression, suggesting that RBM8A might be a potential bio-marker and drug target in HCC therapy.
The RNA Processing Factor Y14 Participates in DNA Damage Response and Repair.
Chuang Tzu-Wei,Lu Chia-Chen,Su Chun-Hao,Wu Pei-Yu,Easwvaran Sarasvathi,Lee Chi-Chieh,Kuo Hung-Che,Hung Kuan-Yang,Lee Kuo-Ming,Tsai Ching-Yen,Tarn Woan-Yuh
DNA repair deficiency leads to genome instability and hence human disease. Depletion of the RNA processing factor Y14/RBM8A in cultured cells or Rbm8a haplodeficiency in the developing mouse cortex results in the accumulation of DNA damage. Y14 depletion differentially affected the expression of DNA damage response (DDR) factors and induced R-loops, both of which threaten genomic stability. Immunoprecipitation coupled with mass spectrometry revealed DDR factors as potential Y14-interacting partners. Further results confirmed that Y14 interacts with Ku and several DDR factors in an ATM-dependent manner. Y14 co-fractionated with Ku in chromatin-enriched fractions and further accumulated on chromatin upon DNA damage. Y14 knockdown delayed recruitment of DDR factors to DNA damage sites and formation of γH2AX foci and also led to Ku retention on chromatin. Accordingly, Y14 depletion compromised the efficiency of DNA end joining. Therefore Y14 likely plays a direct role in DNA damage repair via its interaction with DDR factors.
TAR syndrome: Clinical and molecular characterization of a cohort of 26 patients and description of novel noncoding variants of RBM8A.
Boussion Simon,Escande Fabienne,Jourdain Anne-Sophie,Smol Thomas,Brunelle Perrine,Duhamel Céline,Alembik Yves,Attié-Bitach Tania,Baujat Geneviève,Bazin Anne,Bonnière Maryse,Carassou Philippe,Carles Dominique,Devisme Louise,Goizet Cyril,Goldenberg Alice,Grotto Sarah,Guichet Agnès,Jouk Pierre-Simon,Loeuillet Laurence,Mechler Charlotte,Michot Caroline,Pelluard Fanny,Putoux Audrey,Whalen Sandra,Ghoumid Jamal,Manouvrier-Hanu Sylvie,Petit Florence
Thrombocytopenia-absent radius (TAR) syndrome is characterized by radial defect and neonatal thrombocytopenia. It is caused by biallelic variants of RBM8A gene (1q21.1) with the association of a null allele and a hypomorphic noncoding variant. RBM8A encodes Y14, a core protein of the exon junction complex involved in messenger RNA maturation. To date, only two hypomorphic variants have been identified. We report on a cohort of 26 patients affected with TAR syndrome and carrying biallelic variants in RBM8A. Half patients carried a 1q21.1 deletion and one of the two known hypomorphic variants. Four novel noncoding variants of RBM8A were identified in the remaining patients. We developed experimental models enabling their functional characterization in vitro. Two variants, located respectively in the 5'-untranslated region (5'-UTR) and 3'-UTR regions, are responsible for a diminished expression whereas two intronic variants alter splicing. Our results bring new insights into the molecular knowledge of TAR syndrome and enabled us to propose genetic counseling for patients' families.
Identification and structural analysis of human RBM8A and RBM8B: two highly conserved RNA-binding motif proteins that interact with OVCA1, a candidate tumor suppressor.
Salicioni A M,Xi M,Vanderveer L A,Balsara B,Testa J R,Dunbrack R L,Godwin A K
The OVCA1 gene is a candidate for the breast and ovarian tumor suppressor gene at chromosome 17p13.3. To help determine the function(s) of OVCA1, we used a yeast two-hybrid screening approach to identify OVCA1-associating proteins. One such protein, which we initially referred to as BOV-1 (binder of OVCA1-1) is 173 or 174 amino acids in length and appears to be a new member of a highly conserved RNA-binding motif (RBM) protein family that is highly conserved evolutionarily. Northern blot analysis revealed that BOV-1 is ubiquitously expressed and that three distinct messenger RNA species are expressed, 1-, 3.2-, and 5.8-kb transcripts. The 1-kb transcript is the most abundant and is expressed at high levels in the testis, heart, placenta, spleen, thymus, and lymphocytes. Using fluorescence in situ hybridization and the 5.8-kb complementary DNA probe, we determined that BOV-1 maps to both chromosome 5q13-q14 and chromosome 14q22-q23. Further sequence analysis determined that the gene coding the 1- and the 3.2-kb transcripts (HGMW-approved gene symbol RBM8A) maps to 14q22-q23, whereas a second highly related gene coding for the 5.8-kb transcript resides at chromosome 5q13-q14 (HGMW-approved gene symbol RBM8B). The predicted proteins encoded by RBM8A and RBM8B are identical except that RBM8B is 16 amino acids shorter at its N-terminus. Molecular modeling of the RNA-binding domain of RBM8A and RBM8B, based on homology to the sex-lethal protein of Drosophila, identifies conserved residues in the RBM8 protein family that are likely to contact RNA in a protein-RNA complex. The conservation of sequence and structure through such an evolutionarily divergent group of organisms suggests an important function for the RBM8 family of proteins.
Gene expression profiling for the prediction of lymph node metastasis in patients with cervical cancer.
Kim Tae-Joong,Choi Jung-Joo,Kim Woo Young,Choi Chel Hun,Lee Jeong-Won,Bae Duk-Soo,Son Dae-Soon,Kim Jhingook,Park Byung Kwan,Ahn Geunghwan,Cho Eun Yoon,Kim Byoung-Gie
We investigated whether gene expression profiling of primary cervical tumor tissue could be used to predict lymph node (LN) metastasis and compared this with conventional magnetic resonance imaging. We obtained 43 primary cervical cancer samples (16 with LN metastasis and 27 without LN metastasis) for microarray analysis. A prediction model for LN metastasis from the training set was developed by support vector machine methods using a 10-fold cross-validation. The 'LN prediction model' derived from the signature of 156 distinctive genes (P < 0.01) had a prediction accuracy of 77%. Correlation between mRNA expressions measured by microarray and semiquantitative reverse transcription-polymerase chain reaction was ascertained in four (RBM8A, SDHB, SERPINB13, and gamma-interferon) out of 10 genes. Magnetic resonance imaging showed accuracy (69%) for the prediction of LN metastasis. These results suggest that gene expression profiling allows reliable prediction of LN metastasis in cervical cancer.
Expression and gene regulation network of in hepatocellular carcinoma based on data mining.
Lin Yan,Liang Rong,Qiu Yufen,Lv Yufeng,Zhang Jinyan,Qin Gang,Yuan Chunling,Liu Zhihui,Li Yongqiang,Zou Donghua,Mao Yingwei
RNA binding motif protein 8A (RBM8A) is an RNA binding protein in a core component of the exon junction complex. Abnormal expression is associated with carcinogenesis. We used sequencing data from the Cancer Genome Atlas database and Gene Expression Omnibus, analyzed expression and gene regulation networks in hepatocellular carcinoma (HCC). Expression was analyzed using Oncomine and Gene Expression Profiling Interactive Analysis tools, while alterations and related functional networks were identified using cBioPortal. LinkedOmics was used to identify differential gene expression with and to analyze Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Gene enrichment analysis examined target networks of kinases, miRNAs and transcription factors. We found that is overexpressed and the gene often amplified in HCC. Expression of this gene is linked to functional networks involving the ribosome and RNA metabolic signaling pathways. Functional network analysis suggested that regulates the spliceosome, ribosome, DNA replication and cell cycle signaling via pathways involving several cancer-related kinases, miRNAs and E2F Transcription Factor 1. Our results demonstrate that data mining efficiently reveals information about expression and potential regulatory networks in HCC, laying a foundation for further study of the role of in carcinogenesis.
Immuno-detection of mRNA-binding protein complex in human cells under transmission electron microscopy.
Ma Qingfeng,Tatsuno Takanori,Nakamura Yuka,Izumi Shin-Ichi,Tomosugi Naohisa,Ishigaki Yasuhito
Microscopy research and technique
Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA-protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real-time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA-protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring-derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA-protein complex in human culture cells by using immuno-electron microscopy. Through immuno-detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA-conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.
C-terminal short arginine/serine repeat sequence-dependent regulation of Y14 (RBM8A) localization.
Tatsuno Takanori,Ishigaki Yasuhito
Y14 (RBM8A) is an RNA recognition motif-containing protein that forms heterodimers with MAGOH and serves as a core factor of the RNA surveillance machinery for the exon junction complex (EJC). The role of the Y14 C-terminal serine/arginine (RS) repeat-containing region, which has been reported to undergo modifications such as phosphorylation and methylation, has not been sufficiently investigated. Thus, we aimed to explore the functional significance of the Y14 C-terminal region. Deletion or dephosphorylation mimic mutants of the C-terminal region showed a shift in localization from the nucleoplasmic region; in addition, the C-terminal RS repeat-containing sequence itself exhibited the potential for nucleolar localization. Additionally, the regulation of Y14 localization by the C-terminal region was further found to be exquisitely controlled by MAGOH binding. Cumulatively, our findings, which demonstrated that Y14 localization is regulated not only by the previously reported N-terminal localization signal but also by the C-terminal RS repeat-containing region through phosphorylation and MAGOH binding to Y14, provide new insights for the mechanism of localization of short RS repeat-containing proteins.
Glycogen synthase kinase-3 and alternative splicing.
Liu Xiaolei,Klein Peter S
Wiley interdisciplinary reviews. RNA
Glycogen synthase kinase-3 (GSK-3) is a highly conserved negative regulator of receptor tyrosine kinase, cytokine, and Wnt signaling pathways. Stimulation of these pathways inhibits GSK-3 to modulate diverse downstream effectors that include transcription factors, nutrient sensors, glycogen synthesis, mitochondrial function, circadian rhythm, and cell fate. GSK-3 also regulates alternative splicing in response to T-cell receptor activation, and recent phosphoproteomic studies have revealed that multiple splicing factors and regulators of RNA biosynthesis are phosphorylated in a GSK-3-dependent manner. Furthermore, inhibition of GSK-3 alters the splicing of hundreds of mRNAs, indicating a broad role for GSK-3 in the regulation of RNA processing. GSK-3-regulated phosphoproteins include SF3B1, SRSF2, PSF, RBM8A, nucleophosmin 1 (NPM1), and PHF6, many of which are mutated in leukemia and myelodysplasia. As GSK-3 is inhibited by pathways that are pathologically activated in leukemia and loss of Gsk3 in hematopoietic cells causes a severe myelodysplastic neoplasm in mice, these findings strongly implicate GSK-3 as a critical regulator of mRNA processing in normal and malignant hematopoiesis. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.