BMC Molecular Biology[JOURNAL]
Sun
200 papers
Gene expression profiling of the venom gland from the Venezuelan mapanare (Bothrops colombiensis) using expressed sequence tags (ESTs).
Suntravat M, Uzcategui NL, Atphaisit C … +4 more , Helmke TJ, Lucena SE, Sánchez EE, Acosta AR
BMC Mol Biol · 2016 Mar · PMID 26944950 · Full text
BACKGROUND: Bothrops colombiensis is a highly dangerous pit viper and responsible for over 70% of snakebites in Venezuela. Although the composition in B. colombiensis venom has been identified using a proteome analysis,...
BACKGROUND: Bothrops colombiensis is a highly dangerous pit viper and responsible for over 70% of snakebites in Venezuela. Although the composition in B. colombiensis venom has been identified using a proteome analysis, the venom gland transcriptome is currently lacking. RESULTS: We constructed a cDNA library from the venom gland of B. colombiensis, and a set of 729 high quality expressed sequence tags (ESTs) was identified. A total number of 344 ESTs (47.2% of total ESTs) was related to toxins. The most abundant toxin transcripts were metalloproteinases (37.5%), phospholipases A2s (PLA2, 29.7%), and serine proteinases (11.9%). Minor toxin transcripts were linked to waprins (5.5%), C-type lectins (4.1%), ATPases (2.9%), cysteine-rich secretory proteins (CRISP, 2.3%), snake venom vascular endothelium growth factors (svVEGF, 2.3%), L-amino acid oxidases (2%), and other putative toxins (1.7%). While 160 ESTs (22% of total ESTs) coded for translation proteins, regulatory proteins, ribosomal proteins, elongation factors, release factors, metabolic proteins, and immune response proteins. Other proteins detected in the transcriptome (87 ESTs, 11.9% of total ESTs) were undescribed proteins with unknown functions. The remaining 138 (18.9%) cDNAs had no match with known GenBank accessions. CONCLUSION: This study represents the analysis of transcript expressions and provides a physical resource of unique genes for further study of gene function and the development of novel molecules for medical applications.
Knockdown of SALL4 inhibits the proliferation and reverses the resistance of MCF-7/ADR cells to doxorubicin hydrochloride.
Chen YY, Li ZZ, Ye YY … +6 more , Xu F, Niu RJ, Zhang HC, Zhang YJ, Liu YB, Han BS
BMC Mol Biol · 2016 Mar · PMID 26935744 · Full text
BACKGROUND: Breast cancer is the most frequent malignancy in women and drug resistance is the major obstacle for its successful chemotherapy. In the present study, we analyzed the involvement of an oncofetal gene, sal-li...
BACKGROUND: Breast cancer is the most frequent malignancy in women and drug resistance is the major obstacle for its successful chemotherapy. In the present study, we analyzed the involvement of an oncofetal gene, sal-like 4 (SALL4), in the tumor proliferation and drug resistance of human breast cancer. RESULTS: Our study showed that SALL4 was up-regulated in the drug resistant breast cancer cell line, MCF-7/ADR, compared to the other five cell lines. We established the lentiviral system expressing short hairpin RNA to knockdown SALL4 in MCF-7/ADR cells. Down-regulation of SALL4 inhibited the proliferation of MCF-7/ADR cells and induced the G1 phase arrest in cell cycle, accompanied by an obvious reduction of the expression of cyclinD1 and CDK4. Besides, down-regulating SALL4 can re-sensitize MCF-7/ADR to doxorubicin hydrochloride (ADMh) and had potent synergy with ADMh in MCF-7/ADR cells. Depletion of SALL4 led to a decrease in IC50 for ADMh and an inhibitory effect on the ability to form colonies in MCF-7/ADR cells. With SALL4 knockdown, ADMh accumulation rate of MCF-7/ADR cells was increased, while the expression of BCRP and c-myc was significantly decreased. Furthermore, silencing SALL4 also suppressed the growth of the xenograft tumors and reversed their resistance to ADMh in vivo. CONCLUSION: SALL4 knockdown inhibits the growth of the drug resistant breast cancer due to cell cycle arrest and reverses tumor chemo-resistance through down-regulating the membrane transporter, BCPR. Thus, SALL4 has potential as a novel target for the treatment of breast cancer.
What is normal? Next generation sequencing-driven analysis of the human circulating miRNAOme.
Tonge DP, Gant TW
BMC Mol Biol · 2016 Feb · PMID 26860190 · Full text
BACKGROUND: MicroRNAs (miRNAs) are short non-protein-coding RNA species that have a regulatory function in modulating protein translation and degradation of specific mRNAs. MicroRNAs are estimated to target approximately...
BACKGROUND: MicroRNAs (miRNAs) are short non-protein-coding RNA species that have a regulatory function in modulating protein translation and degradation of specific mRNAs. MicroRNAs are estimated to target approximately 60% of all human mRNAs and are associated with the regulation of all physiological processes. Similar to many messenger RNAs (mRNA), miRNAs exhibit marked tissue specificity, and appear to be dysregulated in response to specific pathological conditions. Perhaps, one of the most significant findings is that miRNAs are detectable in various biological fluids and are stable during routine clinical processing, paving the way for their use as novel biomarkers. Despite an increasing number of publications reporting individual miRNAs or miRNA signatures to be diagnostic of disease or indicative of response to therapy, there is still a paucity of baseline data necessary for their validation. To this end, we utilised state of the art sequencing technologies to determine the global expression of all circulating miRNAs within the plasma of 18 disease-free human subjects. RESULTS: In excess of 500 miRNAs were detected in our study population with expression levels across several orders of magnitude. Ten highly expressed miRNAs accounted for 90% of the total reads that mapped showing that despite the range of miRNAs present, the total miRNA load of the plasma was predominated by just these few species (50% of which are blood cell associated). Ranges of expression were determined for all miRNA detected (>500) and a set of highly stable miRNAs identified. Finally, the effects of gender, smoking status and body mass index on miRNA expression were determined. CONCLUSIONS: The data contained within will be of particular use to researchers performing miRNA-based biomarker screening in plasma and allow shortlisting of candidates a priori to expedite discovery or reduce costs as required.
TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint.
Awate S, De Benedetti A
BMC Mol Biol · 2016 Feb · PMID 26860083 · Full text
BACKGROUND: The Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activ...
BACKGROUND: The Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activity it phosphorylates Rad9 at S328. In this work we investigated the significance of this mechanism by overexpressing mutant TLK1B in which the inhibitory phosphorylation site was eliminated. RESULTS AND DISCUSSION: These cells expressing TLK1B resistant to DNA damage showed constitutive phosphorylation of Rad9 S328 that occurred even in the presence of hydroxyurea (HU), and this resulted in a delayed checkpoint recovery. One possible explanation was that premature phosphorylation of Rad9 caused its dissociation from 9-1-1 at stalled replication forks, resulting in their collapse and prolonged activation of the S-phase checkpoint. We found that phosphorylation of Rad9 at S328 results in its dissociation from chromatin and redistribution to the cytoplasm. This results in double stranded breaks formation with concomitant activation of ATM and phosphorylation of H2AX. Furthermore, a Rad9 (S328D) phosphomimic mutant was exclusively localized to the cytoplasm and not the chromatin. Another Rad9 phosphomimic mutant (T355D), which is also a site phosphorylated by TLK1, localized normally. In cells expressing the mutant TLK1B treated with HU, Rad9 association with Hus1 and WRN was greatly reduced, suggesting again that its phosphorylation causes its premature release from stalled forks. CONCLUSIONS: We propose that normally, the inactivation of TLK1B following replication arrest and genotoxic stress functions to allow the retention of 9-1-1 at the sites of damage or stalled forks. Following reactivation of TLK1B, whose synthesis is concomitantly induced by genotoxins, Rad9 is hyperphosphorylated at S328, resulting in its dissociation and inactivation of the checkpoint that occurs once repair is complete.
Erratum to: Temperature-induced variation in gene expression burst size in metazoan cells.
Arnaud O, Meyer S, Vallin E … +2 more , Beslon G, Gandrillon O
BMC Mol Biol · 2016 Feb · PMID 26841723 · Full text
Abstract loading — click title to view on PubMed.
Structural and functional analysis of four non-coding Y RNAs from Chinese hamster cells: identification, molecular dynamics simulations and DNA replication initiation assays.
de Lima Neto QA, Duarte Junior FF, Bueno PS … +5 more , Seixas FA, Kowalski MP, Kheir E, Krude T, Fernandez MA
BMC Mol Biol · 2016 Jan · PMID 26733090 · Full text
BACKGROUND: The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no informa...
BACKGROUND: The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no information is available about Y RNAs in Chinese hamster cells, which have already been used to detect replication origins and alternative DNA structures around these sites. Here, we report the gene sequences and predicted structural characteristics of the Chinese hamster Y RNAs, and analyze their ability to support the initiation of chromosomal DNA replication in vitro. RESULTS: We identified DNA sequences in the Chinese hamster genome of four Y RNAs (chY1, chY3, chY4 and chY5) with upstream promoter sequences, which are homologous to the four main types of vertebrate Y RNAs. The chY1, chY3 and chY5 genes were highly conserved with their vertebrate counterparts, whilst the chY4 gene showed a relatively high degree of diversification from the other vertebrate Y4 genes. Molecular dynamics simulations suggest that chY4 RNA is structurally stable despite its evolutionarily divergent predicted stem structure. Of the four Y RNA genes present in the hamster genome, we found that only the chY1 and chY3 RNA were strongly expressed in the Chinese hamster GMA32 cell line, while expression of the chY4 and chY5 RNA genes was five orders of magnitude lower, suggesting that they may in fact not be expressed. We synthesized all four chY RNAs and showed that any of these four could support the initiation of DNA replication in an established human cell-free system. CONCLUSIONS: These data therefore establish that non-coding chY RNAs are stable structures and can substitute for human Y RNAs in a reconstituted cell-free DNA replication initiation system. The pattern of Y RNA expression and functionality is consistent with Y RNAs of other rodents, including mouse and rat.
microRNA-150 promotes cervical cancer cell growth and survival by targeting FOXO4.
Li J, Hu L, Tian C … +3 more , Lu F, Wu J, Liu L
BMC Mol Biol · 2015 Dec · PMID 26715362 · Full text
BACKGROUND: Dysregulation of microRNA-150 (miR-150) is commonly observed in solid tumor and has been reported to be involved in multiple important biological processes, such as cell proliferation, apoptosis, and metastas...
BACKGROUND: Dysregulation of microRNA-150 (miR-150) is commonly observed in solid tumor and has been reported to be involved in multiple important biological processes, such as cell proliferation, apoptosis, and metastasis. Elevated miR-150 level was also detected in cervical carcinoma, whereas its function in cancer progression has not been studied yet. METHODS: The expression of miRNA-150 in cervical carcinoma was compared with normal cervical tissue and using qRT-PCR. The effects of miR-150 on cell cycle and apoptosis, as well as the expression of cycle- and apoptosis-related genes, were determined using flow cytometry, TUNEL assay, qRT-PCR, and Western blot, respectively. The direct target of miR-150 was confirmed using 3' untranslated region (UTR) luciferase reporter assay. RESULTS: miR-150 promotes cervical cancer cell survival and growth, while the inhibition of miR-150 suppresses these actions. miR-150 also induced the cell cycle progression from G1/G0 to S phase, resulting in an enhancement of growth. Several cell cycle- and apoptosis-related genes, CyclinD1, p27, BIM, and FASL were modulated by miR-150. Moreover, miR-150 directly reduced the expression of FOXO4, which regulates the expression of CyclinD1, p27, BIM, and FASL, by targeting its 3' UTR. CONCLUSION: Taken together, our data demonstrated that elevated miR-150 targets FOXO4 expression and therefore regulates multiple genes expression, resulting in cervical cancer cell growth and survival.
Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.
Fietz D, Markmann M, Lang D … +6 more , Konrad L, Geyer J, Kliesch S, Chakraborty T, Hossain H, Bergmann M
BMC Mol Biol · 2015 Dec · PMID 26715186 · Full text
BACKGROUND: Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is...
BACKGROUND: Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR. RESULTS: Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes. CONCLUSION: We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.
Conserved and highly expressed tRNA derived fragments in zebrafish.
Soares AR, Fernandes N, Reverendo M … +4 more , Araújo HR, Oliveira JL, Moura GM, Santos MA
BMC Mol Biol · 2015 Dec · PMID 26694924 · Full text
BACKGROUND: Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identif...
BACKGROUND: Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identification by next generation sequencing (NGS) we are still far from understanding their full diversity and functional repertoire. RESULTS: Here we report the identification of tRNA derived fragments (tRFs) by NGS of the sncRNA fraction of zebrafish. The tRFs identified are 18-30 nt long, are derived from specific 5' and 3' processing of mature tRNAs and are differentially expressed during development and in differentiated tissues, suggesting that they are likely produced by specific processing rather than random degradation of tRNAs. We further show that a highly expressed tRF (5'tRF-Pro(CGG)) is cleaved in vitro by Dicer and has silencing ability, indicating that it can enter the RNAi pathway. A computational analysis of zebrafish tRFs shows that they are conserved among vertebrates and mining of publicly available datasets reveals that some 5'tRFs are differentially expressed in disease conditions, namely during infection and colorectal cancer. CONCLUSIONS: tRFs constitute a class of conserved regulatory RNAs in vertebrates and may be involved in mechanisms of genome regulation and in some diseases.
Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells.
Lu Y, Li J, Cheng J … +1 more , Lubahn DB
BMC Mol Biol · 2015 Dec · PMID 26627478 · Full text
BACKGROUND: Orphan nuclear receptor estrogen related receptor β (Esrrb or ERRβ) is well known in stem cells and early embryonic development. However, little is known about its function in cancer. METHOD: We investigated...
BACKGROUND: Orphan nuclear receptor estrogen related receptor β (Esrrb or ERRβ) is well known in stem cells and early embryonic development. However, little is known about its function in cancer. METHOD: We investigated the mRNA profile alterations induced by Esrrb expression and its synthetic ligand DY131 in human prostate cancer DU145 cells via RNA-Seq analysis. RESULTS: We distinguished 67 mRNAs differentially expressed by Esrrb alone. Although DY131 alone did not change any gene, treatment of DY131 in the presence of Esrrb altered 1161 mRNAs. These observations indicated Esrrb had both ligand-independent and ligand-dependent activity. When Esrrb was expressed, DY131 treatment further regulated 15 Esrrb-altered mRNAs. DY131 acted as an antagonist for 11 of 15 mRNAs (wdr52, f13a1, pxdn, spns2, loc100506599, tagln, loc441454, tkel1, sema3f, zcwpw2, sdc2) and as an agonist for 4 of the 15 mRNAs (rarres3, oasl, padi2, ddx60). Gene ontology analyses showed altered genes are related to transcription and translation regulation, cell proliferation and apoptosis regulation, and cellular metabolism. CONCLUSION: Our results characterized mRNA profiles in DU145 prostate cancer cells driven by Esrrb expression and Esrrb ligand DY131, and provided multiple markers to characterize Esrrb's function in Esrrb research.
Temperature-induced variation in gene expression burst size in metazoan cells.
Arnaud O, Meyer S, Vallin E … +2 more , Beslon G, Gandrillon O
BMC Mol Biol · 2015 Nov · PMID 26608344 · Full text
BACKGROUND: Gene expression is an inherently stochastic process, owing to its dynamic molecular nature. Protein amount distributions, which can be acquired by cytometry using a reporter gene, can inform about the mechani...
BACKGROUND: Gene expression is an inherently stochastic process, owing to its dynamic molecular nature. Protein amount distributions, which can be acquired by cytometry using a reporter gene, can inform about the mechanisms of the underlying microscopic molecular system. RESULTS: By using different clones of chicken erythroid progenitor cells harboring different integration sites of a CMV-driven mCherry protein, we investigated the dynamical behavior of such distributions. We show that, on short term, clone distributions can be quickly regenerated from small population samples with a high accuracy. On longer term, on the contrary, we show variations manifested by correlated fluctuation in the Mean Fluorescence Intensity. In search for a possible cause of this correlation, we demonstrate that in response to small temperature variations cells are able to adjust their gene expression rate: a modest (2 °C) increase in external temperature induces a significant down regulation of mean expression values, with a reverse effect observed when the temperature is decreased. Using a two-state model of gene expression we further demonstrate that temperature acts by modifying the size of transcription bursts, while the burst frequency of the investigated promoter is less systematically affected. CONCLUSIONS: For the first time, we report that transcription burst size is a key parameter for gene expression that metazoan cells from homeotherm animals can modify in response to an external thermal stimulus.
Genes targeted by the Hedgehog-signaling pathway can be regulated by Estrogen related receptor β.
Lu Y, Li J, Cheng J … +1 more , Lubahn DB
BMC Mol Biol · 2015 Nov · PMID 26597826 · Full text
BACKGROUND: Nuclear receptor family member, Estrogen related receptor β, and the Hedgehog signal transduction pathway are both reported to relate to tumorigenesis and induced pluripotent stem cell reprogramming. We hypot...
BACKGROUND: Nuclear receptor family member, Estrogen related receptor β, and the Hedgehog signal transduction pathway are both reported to relate to tumorigenesis and induced pluripotent stem cell reprogramming. We hypothesize that Estrogen related receptor β can modulate the Hedgehog signaling pathway and affect Hedgehog driven downstream gene expression. RESULTS: We established an estrogen related receptor β-expressing Hedgehog-responsive NIH3T3 cell line by Esrrb transfection, and performed mRNA profiling using RNA-Seq after Hedgehog ligand conditioned medium treatment. Esrrb expression altered 171 genes, while Hedgehog signaling activation alone altered 339 genes. Additionally, estrogen related receptor β expression in combination with Hedgehog signaling activation affects a group of 109 Hedgehog responsive mRNAs, including Hsd11b1, Ogn, Smoc2, Igf1, Pdcd4, Igfbp4, Stmn1, Hp, Hoxd8, Top2a, Tubb4b, Sfrp2, Saa3, Prl2c3 and Dpt. CONCLUSIONS: We conclude that Estrogen related receptor β is capable of interacting with Hh-signaling downstream targets. Our results suggest a new level of regulation of Hedgehog signaling by Estrogen related receptor β, and indicate modulation of Estrogen related receptor β can be a new strategy to regulate various functions driven by the Hedgehog signaling pathway.
DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts.
Solovjeva L, Firsanov D, Vasilishina A … +4 more , Chagin V, Pleskach N, Kropotov A, Svetlova M
BMC Mol Biol · 2015 Oct · PMID 26458748 · Full text
BACKGROUND: Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after se...
BACKGROUND: Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as "culture stress". Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs). RESULTS: The effect of the drug was estimated by immunoblotting and immunofluorescence microscopy using the antibody to phosphorylated histone H2AX (gH2AX), which is generally accepted as a DSB marker. At all stages of the cell cycle, both presenescent and young cells demonstrated variability of the number of gH2AX foci per nucleus. gH2AX focus induction was found to be independent from BL-hydrolase expression. Some differences in DSB repair process between BL-treated young and presenescent Syrian hamster cells were observed: (1) the kinetics of gH2AX focus loss in G0 fibroblasts of young culture was faster than in cells that prematurely stopped dividing; (2) presenescent cells were characterized by a slower recruitment of DSB repair proteins 53BP1, phospho-DNA-PK and phospho-ATM to gH2AX focal sites, while the rate of phosphorylated ATM/ATR substrate accumulation was the same as that in young cells. CONCLUSIONS: Our results demonstrate an impairment of DSB repair in prematurely aged Syrian hamster fibroblasts in comparison with young fibroblasts, suggesting age-related differences in response to BL therapy.
The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner.
Samra N, Atir-Lande A, Pnueli L … +1 more , Arava Y
BMC Mol Biol · 2015 Sep · PMID 26404137 · Full text
BACKGROUND: mRNA binding proteins (RBPs) constitute 10-15% of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature...
BACKGROUND: mRNA binding proteins (RBPs) constitute 10-15% of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature its interaction with RBPs, identification of novel RBPs is a significant challenge. Recently, a novel methodology for RBP purification and identification (termed RaPID) was presented, which allows high affinity purification of RBPs while associated with mRNA in vivo. RESULTS: We performed a RaPID screen for proteins that interact with PMP1 mRNA in order to identify novel mRNA binding proteins. PMP1 mRNA was tagged in its 3' UTR with multiple MS2 loops and co-expressed with MS2-binding protein fused to streptavidin binding protein (SBP). RNA-protein complexes were cross-linked in vivo and isolated through streptavidin beads. The eluted proteins were subjected to mass spectroscopy analysis. The screen identified many proteins, about half of them were previously shown to bind RNA. We focused on eEF3 (YEF3), an essential translation elongation factor that interacts with ribosomes. Purification of TAP-tagged Yef3 with its associated RNAs confirmed that the native PMP1 transcript is associated with it. Intriguingly, high association with Yef3-TAP was observed when purification was performed in the presence of EDTA, and with PMP1 that contains stop codons immediately downstream to the initiation codon. Furthermore, high association was observed with a transcript containing only the 3' UTR of PMP1. Complementary, RaPID isolation of MS2-tagged 3' UTRs with their associated proteins revealed that Yef3 can efficiently interact with these regions. CONCLUSIONS: This study identifies many novel proteins that interact with PMP1 mRNA. Importantly, the elongation factor Yef3 was found to interact with mRNA in non-coding regions and in a translation independent manner. These results suggest an additional, non-elongation function for this factor.
Deciphering targeting rules of splicing modulator compounds: case of TG003.
Sakuma M, Iida K, Hagiwara M
BMC Mol Biol · 2015 Sep · PMID 26400733 · Full text
BACKGROUND: Recent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. Splicing-targeting therapy tries to ameliorate the...
BACKGROUND: Recent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. Splicing-targeting therapy tries to ameliorate the disease by altering the exon combination of transcripts to reduce the undesired effect of genetic mutations. However, the knowledge and tools to understand factors contributing to splicing modulator compound sensitivity have been lacking. Our goal was to establish a method to characterize sequence features found in compound sensitive exons. RESULTS: Here we developed a comparative transcriptomic approach to explore features that make an exon sensitive to a chemical compound. In this study, we chose TG003, a potential drug for Duchenne muscular dystrophy, and performed RNA-sequencing on samples from human and mouse skeletal muscle cells, with and without TG003 treatments. We compared TG003 responsiveness between homologous exon pairs and identified 21 pairs in which human exons were skip-enhanced but not mouse exons. We compared the sequence features; splice site scores, number of splicing factor binding sites, and properties of branch sequence and polypyrimidine tracts, and found that polypyrimidine tracts were stronger (longer stretches and richer content of consecutive polypyrimidine) in the mouse TG003 insensitive exons. We also compared the features between TG003 skip-enhanced and insensitive exons within the species, and discovered that human TG003 skip-enhanced exons were shorter and had less splicing factor binding sites than the group of human TG003 insensitive exons. Mouse insensitive exons homologous to human TG003 skip-enhanced exons shared these properties. Our results suggested that these features are prerequisites for TG003 skip-enhanced exons and weak polypyrimidine tracts are defining features, which were supported by a decision tree analysis on all cassette exons in human. CONCLUSIONS: In this study we established a comparative transcriptomic approach, which shed lights on how small chemical compounds modulate RNA splicing. The results described here was the first attempt to decipher the targeting rules of a splicing modulator compound. We expect that this approach would contribute to the precise understanding of the mechanism of TG003-induced splicing modulation, expand target diseases of splicing modulators in general, as well as the development of new splicing modulators.
RE1 silencing transcription factor (REST) negatively regulates ALL1-fused from chromosome 1q (AF1q) gene transcription.
Hu Y, Sun Q, Zhang C … +2 more , Sha Q, Sun X
BMC Mol Biol · 2015 Sep · PMID 26341630 · Full text
BACKGROUND: ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in neuronal development; however, its upstream regulatory mechanisms in neural system remained elusive....
BACKGROUND: ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in neuronal development; however, its upstream regulatory mechanisms in neural system remained elusive. RESULTS: Our study showed that REST (RE1 silencing transcription factor), a key transcription factor in neurodevelopment, could down-regulate the gene expression of AF1q. The promoter assay identified a neuron-restrictive silencer element at -383 to -363 bp of human AF1q promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (CHIP) confirmed the binding of REST to the NRSE in AF1q gene promoter. Additionally, the negative correlation between the expression levels of Af1q and Rest in mice neurodevelopment supported the negative regulation of AF1q by REST and the potential functions of AF1q in neurodevelopment. CONCLUSION: These results demonstrate that REST regulates AF1q gene transcription through directly binding to a NRSE at -383 to -363 bp of AF1q promoter.
TRIM28 as a novel transcriptional elongation factor.
Bunch H, Calderwood SK
BMC Mol Biol · 2015 Aug · PMID 26293668 · Full text
TRIM28 is a multidomain protein with versatile functions in transcription and DNA repair. Recently it was shown that this factor plays unanticipated roles in transcriptional elongation. TRIM28 was shown to stabilize the...
TRIM28 is a multidomain protein with versatile functions in transcription and DNA repair. Recently it was shown that this factor plays unanticipated roles in transcriptional elongation. TRIM28 was shown to stabilize the pausing of RNA polymerase II (Pol II) close to the transcriptional start site in many unactivated genes, permitting Pol II accumulation and readying genes for induction. In addition, the factor was shown to respond rapidly to signals accompanying transcriptional activation permitting the productive elongation of RNA by previously paused Pol II. We discuss here critical regulatory mechanisms of TRIM28 in transcriptional control and DNA repair that may illuminate the novel roles of this factor in pausing and elongation of Pol II.
Analysis artefacts of the INS-IGF2 fusion transcript.
Wernersson R, Frogne T, Rescan C … +5 more , Hansson L, Bruun C, Grønborg M, Jensen JN, Madsen OD
BMC Mol Biol · 2015 Jul · PMID 26220792 · Full text
BACKGROUND: In gene expression analysis, overlapping genes, splice variants, and fusion transcripts are potential sources of data analysis artefacts, depending on how the observed intensity is assigned to one, or more ge...
BACKGROUND: In gene expression analysis, overlapping genes, splice variants, and fusion transcripts are potential sources of data analysis artefacts, depending on how the observed intensity is assigned to one, or more genes. We here exemplify this by an in-depth analysis of the INS-IGF2 fusion transcript, which has recently been reported to be among the highest expressed transcripts in human pancreatic beta cells and its protein indicated as a novel autoantigen in Type 1 Diabetes. RESULTS: Through RNA sequencing and variant specific qPCR analyses we demonstrate that the true abundance of INS-IGF2 is >20,000 fold lower than INS in human beta cells, and we suggest an explanation to the nature of the artefacts which have previously led to overestimation of the gene expression level in selected studies. We reinvestigated the previous reported findings of detection of INS-IGF2 using antibodies both in Western blotting and immunohistochemistry. We found that the one available commercial antibody (BO1P) raised against recombinant INS-IGF2 show strong cross-reaction to native proinsulin, and we did not detect INS-IGF2 protein in the human beta cell line EndoC-βH1. Furthermore, using highly sensitive proteomics analysis we could not demonstrate INS-IGF2 protein in samples of human islets nor in EndoC-βH1. CONCLUSIONS: Sequence features, such as fusion transcripts spanning multiple genes can lead to unexpected results in gene expression analysis, and care must be taken in generating and interpreting the results. For the specific case of INS-IGF2 we conclude that the abundance of the fusion transcript/protein is exceedingly lower than previously reported, and that current immuno-reagents available for detecting INS-IGF2 protein have a strong cross-reaction to native human proinsulin. Finally, we were unable to detect INS-IGF2 protein by proteomics analysis.
SIRT6 protein deacetylase interacts with MYH DNA glycosylase, APE1 endonuclease, and Rad9-Rad1-Hus1 checkpoint clamp.
Hwang BJ, Jin J, Gao Y … +8 more , Shi G, Madabushi A, Yan A, Guan X, Zalzman M, Nakajima S, Lan L, Lu AL
BMC Mol Biol · 2015 Jun · PMID 26063178 · Full text
BACKGROUND: SIRT6, a member of the NAD(+)-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved...
BACKGROUND: SIRT6, a member of the NAD(+)-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved in mutation avoidance from oxidative DNA damage. Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp promotes cell cycle checkpoint signaling and DNA repair. BER is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. SIRT6 is involved in DNA double-strand break repair and has been implicated in BER. Here we investigate the direct physical and functional interactions between SIRT6 and BER enzymes. RESULTS: We show that SIRT6 interacts with and stimulates MYH glycosylase and APE1. In addition, SIRT6 interacts with the 9-1-1 checkpoint clamp. These interactions are enhanced following oxidative stress. The interdomain connector of MYH is important for interactions with SIRT6, APE1, and 9-1-1. Mutagenesis studies indicate that SIRT6, APE1, and Hus1 bind overlapping but different sequence motifs on MYH. However, there is no competition of APE1, Hus1, or SIRT6 binding to MYH. Rather, one MYH partner enhances the association of the other two partners to MYH. Moreover, APE1 and Hus1 act together to stabilize the MYH/SIRT6 complex. Within human cells, MYH and SIRT6 are efficiently recruited to confined oxidative DNA damage sites within transcriptionally active chromatin, but not within repressive chromatin. In addition, Myh foci induced by oxidative stress and Sirt6 depletion are frequently localized on mouse telomeres. CONCLUSIONS: Although SIRT6, APE1, and 9-1-1 bind to the interdomain connector of MYH, they do not compete for MYH association. Our findings indicate that SIRT6 forms a complex with MYH, APE1, and 9-1-1 to maintain genomic and telomeric integrity in mammalian cells.
MicroRNA-19a regulates lipopolysaccharide-induced endothelial cell apoptosis through modulation of apoptosis signal-regulating kinase 1 expression.
Jiang WL, Zhang YF, Xia QQ … +4 more , Zhu J, Yu X, Fan T, Wang F
BMC Mol Biol · 2015 May · PMID 25982447 · Full text
BACKGROUND: MicroRNAs, small non-encoding RNAs that post-transcriptionally modulate expression of their target genes, have been implicated as critical regulatory molecules in endothelial cells. RESULTS: In the present st...
BACKGROUND: MicroRNAs, small non-encoding RNAs that post-transcriptionally modulate expression of their target genes, have been implicated as critical regulatory molecules in endothelial cells. RESULTS: In the present study, we found that overexpression of miR-19a protects endothelial cells from lipopolysaccharide (LPS)-induced apoptosis through the apoptosis signal-regulating kinase 1 (ASK1)/p38 pathway. Quantitative real-time PCR demonstrated that the expression of miR-19a in endothelial cell was markedly down-regulated by LPS stimulation. Furthermore, LPS-induced apoptosis was significantly inhibited by over-expression of miR-19a. Finally, both a luciferase reporter assay and western blot analysis showed that ASK1 is a direct target of miR-19a. CONCLUSIONS: MiR-19a regulates ASK1 expression by targeting specific binding sites in the 3' untranslated region of ASK1 mRNA. Overexpression of miR-19a is an effective method to protect against LPS-induced apoptosis of endothelial cells.
About
- Frequency
- Sun
- Papers found
- 200
- RSS feed
- Subscribe