Publications by authors named "Yilei Liu"

21 Publications

  • Page 1 of 1

Regulation of ferroptosis by non‑coding RNAs in the development and treatment of cancer (Review).

Oncol Rep 2021 Jan 5;45(1):29-48. Epub 2020 Nov 5.

Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan 610513, P.R. China.

Ferroptosis, a relatively recently discovered type of cell death that is iron dependent and nonapoptotic, is involved in the accumulation of lipid reactive oxygen species (ROS), and has been shown to serve a vital role in various pathological processes, including those underlying neurodegeneration, ischemic reperfusion injury, acute organ injury, and in particular, tumor biology. Emerging evidence has highlighted the roles of ferroptosis in the development and resistance to chemoradiotherapy in cancer. Recently, an increasing number of studies have shown that non‑coding RNAs modulate the process of ferroptotic cell death, and this has further highlighted the potential of regulation of ferroptosis as a means of cancer management. Although these studies have highlighted the critical role of ferroptosis in cancer therapeutics, the roles of ferroptosis induced by non‑coding RNAs in cancer development remain unclear. Herein, the current body of knowledge of ferroptosis in cancer is summarized and an overview of the mechanisms of ferroptosis and the functions of non‑coding RNAs in regulating ferroptotic cell death are discussed. The future status of ferroptosis in cancer management is deliberated and strategies for treatment of therapy‑resistant cancers are discussed.
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http://dx.doi.org/10.3892/or.2020.7836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709825PMC
January 2021

The Exopolysaccharide Cepacian Plays a Role in the Establishment of the - Symbiosis.

Front Microbiol 2020 16;11:1600. Epub 2020 Jul 16.

Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland.

is a rhizobial strain that belongs to the beta-proteobacteria, a group known to form efficient nitrogen-fixing symbioses within root nodules of several legumes, including the agriculturally important common bean. The establishment of the symbiosis requires the exchange of rhizobial and plant signals such as lipochitooligosaccharides (Nod factors), polysaccharides, and flavonoids. Inspection of the genome of the competitive rhizobium revealed the presence of several polysaccharide biosynthetic gene clusters. In this study, we demonstrate that , a gene encoding a GDP-D-mannose 4,6-dehydratase, which is involved in the production of the exopolysaccharide cepacian, an important component of biofilms produced by closely related opportunistic pathogens of the complex (), is required for efficient plant colonization. Wild-type was shown to produce cepacian while a mutant did not. Additionally, the mutant produced a significantly lower amount of biofilm and formed less root nodules compared to the wild-type strain with as host plant. Finally, expression of the operon containing was induced by the presence of germinated seeds under nitrogen limiting conditions suggesting a role of this polysaccharide in the establishment of this ecologically important symbiosis.
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http://dx.doi.org/10.3389/fmicb.2020.01600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378592PMC
July 2020

A Synthetic Carrier of Nucleic Acids Structured as a Neutral Phospholipid Envelope Tightly Assembled on Polyplex Surface.

Adv Healthc Mater 2020 03 24;9(6):e1901705. Epub 2020 Jan 24.

Center for BioDelivery Sciences, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Rd, Shanghai, 200240, P. R. China.

Synthetic carriers of nucleic acids remain inefficient for practical applications due to their insufficient functions as compared with viral vectors developed by evolution. Here, a synthetic carrier is designed to structurally mimic lentivirus, a widely-used viral vector in therapeutic developments, for its neutral phospholipid membrane tightly anchored on the surface of a packed nucleic acid core. Unlike the reported lipopolyplexes of which the surface membrane around the nucleic acid core is formed from charged lipids, the stable attachment of the neutral lipids to each polyplex core in the present system is achieved through preadsorbed micelles of multicarboxyl amphiphilic molecules as lipid bilayer anchors. The adsorbed micelles are under a tension of deformation due to the electrostatic attraction of the head groups to the cationic surface and their "thermodynamic responsibility" to cover the hydrophobic tails in water. When liposomes of neutral phospholipids approach, the hydrophobic tail groups of the adsorbed micelles may insert into the lipid bilayer matrix to induce them to fuse around polyplex and relieve the thermodynamic tension. The formed neutral phospholipid membrane may encapsulate the polyplex core stably, prevent siRNA from prephagocytic leaking and degrading, and immobilize functional agents with increased capacity.
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http://dx.doi.org/10.1002/adhm.201901705DOI Listing
March 2020

An ancient germ cell-specific RNA-binding protein protects the germline from cryptic splice site poisoning.

Elife 2019 01 24;8. Epub 2019 Jan 24.

Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom.

Male germ cells of all placental mammals express an ancient nuclear RNA binding protein of unknown function called RBMXL2. Here we find that deletion of the retrogene encoding RBMXL2 blocks spermatogenesis. Transcriptome analyses of age-matched deletion mice show that RBMXL2 controls splicing patterns during meiosis. In particular, RBMXL2 represses the selection of aberrant splice sites and the insertion of cryptic and premature terminal exons. Our data suggest a retrogene has been conserved across mammals as part of a splicing control mechanism that is fundamentally important to germ cell biology. We propose that this mechanism is essential to meiosis because it buffers the high ambient concentrations of splicing activators, thereby preventing poisoning of key transcripts and disruption to gene expression by aberrant splice site selection.
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http://dx.doi.org/10.7554/eLife.39304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345566PMC
January 2019

Metabolomics and Transcriptomics Identify Multiple Downstream Targets of Paraburkholderia phymatum σ During Symbiosis with Phaseolus vulgaris.

Int J Mol Sci 2018 Apr 1;19(4). Epub 2018 Apr 1.

Department of Plant and Microbial Biology, University of Zurich, CH-8057 Zurich, Switzerland.

RpoN (or σ) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include α- and β-rhizobia. Our previous studies showed that an mutant of the β-rhizobial strain STM815 formed root nodules on cv. Negro jamapa, which were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of , transcriptomics was combined with a powerful metabolomics approach. The metabolome of root nodules infected by a Fix mutant revealed statistically significant metabolic changes compared to wild-type Fix⁺ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix and Fix⁺ nodules-combined with a search for RpoN binding sequences in promoter regions of regulated genes-confirmed the expected control of σ on nitrogen fixation genes in nodules. The transcriptomic data also allowed us to identify additional target genes, whose differential expression was able to explain the observed metabolite changes in numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the mutant, and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype, as fewer but heavier nodules were formed on roots.
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http://dx.doi.org/10.3390/ijms19041049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979394PMC
April 2018

Transcriptome Analysis of Paraburkholderia phymatum under Nitrogen Starvation and during Symbiosis with Phaseolus Vulgaris.

Genes (Basel) 2017 Dec 15;8(12). Epub 2017 Dec 15.

Department of Plant and Microbial Biology, University of Zurich, CH-8057 Zurich, Switzerland.

belongs to the β-subclass of proteobacteria. It has recently been shown to be able to nodulate and fix nitrogen in symbiosis with several mimosoid and papilionoid legumes. In contrast to the symbiosis of legumes with α-proteobacteria, very little is known about the molecular determinants underlying the successful establishment of this mutualistic relationship with β-proteobacteria. In this study, we performed an RNA-sequencing (RNA-seq) analysis of free-living growing under nitrogen-replete and -limited conditions, the latter partially mimicking the situation in nitrogen-deprived soils. Among the genes upregulated under nitrogen limitation, we found genes involved in exopolysaccharides production and in motility, two traits relevant for plant root infection. Next, RNA-seq data of grown under free-living conditions and from symbiotic root nodules of (common bean) were generated and compared. Among the genes highly upregulated during symbiosis, we identified-besides the gene cluster-an operon encoding a potential cytochrome o ubiquinol oxidase (Bphy_3646-49). Bean root nodules induced by a mutant strain showed reduced nitrogenase and nitrogen fixation abilities, suggesting an important role of the cytochrome for respiration inside the nodule. The analysis of mutant strains for the RNA polymerase transcription factor RpoN (σ) and its activator NifA indicated that-similar to the situation in α-rhizobia- RpoN and NifA are key regulators during symbiosis with .
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http://dx.doi.org/10.3390/genes8120389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748707PMC
December 2017

Identification of AHL- and BDSF-Controlled Proteins in Burkholderia cenocepacia by Proteomics.

Methods Mol Biol 2018 ;1673:193-202

Department of Plant and Microbial Biology, University of Zurich, Zollikerstrasse 107, 8008, Zurich, Switzerland.

We used comparative proteome analysis to determine the target genes of the two quorum sensing (QS) circuits in the opportunistic pathogen Burkholderia cenocepacia: the N-acyl homoserine lactone (AHL)-based CepIR system and the BDSF (B urkholderia diffusible signal factor, cis-2-dodecenoic acid)-based RpfFR system. In this book chapter, we focus on the description of the practical procedure we currently use in the laboratory to perform a sensitive GeLC-MS/MS shotgun proteomics experiment; we also briefly describe the downstream bioinformatic data analysis.
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http://dx.doi.org/10.1007/978-1-4939-7309-5_15DOI Listing
June 2018

NtrC-dependent control of exopolysaccharide synthesis and motility in Burkholderia cenocepacia H111.

PLoS One 2017 29;12(6):e0180362. Epub 2017 Jun 29.

Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland.

Burkholderia cenocepacia is a versatile opportunistic pathogen that survives in a wide variety of environments, which can be limited in nutrients such as nitrogen. We have previously shown that the sigma factor σ54 is involved in the control of nitrogen assimilation and virulence in B. cenocepacia H111. In this work, we investigated the role of the σ54 enhancer binding protein NtrC in response to nitrogen limitation and in the pathogenicity of H111. Of 95 alternative nitrogen sources tested the ntrC showed defects in the utilisation of nitrate, urea, L-citrulline, acetamide, DL-lactamide, allantoin and parabanic acid. RNA-Seq and phenotypic analyses of an ntrC mutant strain showed that NtrC positively regulates two important phenotypic traits: exopolysaccharide (EPS) production and motility. However, the ntrC mutant was not attenuated in C. elegans virulence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180362PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491218PMC
October 2017

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68.

Nat Commun 2016 Jan 13;7:10355. Epub 2016 Jan 13.

Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK.

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.
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http://dx.doi.org/10.1038/ncomms10355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735526PMC
January 2016

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development.

Basic Res Cardiol 2014 Jan 27;109(1):399. Epub 2013 Dec 27.

Division of Cardiology, Department of Medicine, University Hospital Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland,

Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.
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http://dx.doi.org/10.1007/s00395-013-0399-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898152PMC
January 2014

The tissue-specific RNA binding protein T-STAR controls regional splicing patterns of neurexin pre-mRNAs in the brain.

PLoS Genet 2013 Apr 25;9(4):e1003474. Epub 2013 Apr 25.

Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

The RNA binding protein T-STAR was created following a gene triplication 520-610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain.
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http://dx.doi.org/10.1371/journal.pgen.1003474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636136PMC
April 2013

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.

PLoS Genet 2011 Dec 15;7(12):e1002390. Epub 2011 Dec 15.

Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom.

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
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http://dx.doi.org/10.1371/journal.pgen.1002390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240583PMC
December 2011

Post-transcriptional exon shuffling events in humans can be evolutionarily conserved and abundant.

Genome Res 2011 Nov 23;21(11):1788-99. Epub 2011 Sep 23.

Institute of Genetic Medicine, Newcastle University, Newcastle NE1 3BZ, United Kingdom.

In silico analyses have established that transcripts from some genes can be processed into RNAs with rearranged exon order relative to genomic structure (post-transcriptional exon shuffling, or PTES). Although known to contribute to transcriptome diversity in some species, to date the structure, distribution, abundance, and functional significance of human PTES transcripts remains largely unknown. Here, using high-throughput transcriptome sequencing, we identify 205 putative human PTES products from 176 genes. We validate 72 out of 112 products analyzed using RT-PCR, and identify additional PTES products structurally related to 61% of validated targets. Sequencing of these additional products reveals GT-AG dinucleotides at >95% of the splice junctions, confirming that they are processed by the spliceosome. We show that most PTES transcripts are expressed in a wide variety of human tissues, that they can be polyadenylated, and that some are conserved in mouse. We also show that they can extend into 5' and 3' UTRs, consistent with formation via trans-splicing of independent pre-mRNA molecules. Finally, we use real-time PCR to compare the abundance of PTES exon junctions relative to canonical exon junctions within the transcripts from seven genes. PTES exon junctions are present at <0.01% to >90% of the levels of canonical junctions, with transcripts from MAN1A2, PHC3, TLE4, and CDK13 exhibiting the highest levels. This is the first systematic experimental analysis of PTES in human, and it suggests both that the phenomenon is much more widespread than previously thought and that some PTES transcripts could be functional.
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http://dx.doi.org/10.1101/gr.116442.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205564PMC
November 2011

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients.

EMBO J 2010 Apr 25;29(7):1248-61. Epub 2010 Feb 25.

Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55-200 CGG repeats in the 5'-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.
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http://dx.doi.org/10.1038/emboj.2010.21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857464PMC
April 2010

Coupling genetics and post-genomic approaches to decipher the cellular splicing code at a systems-wide level.

Biochem Soc Trans 2010 Feb;38(Pt 1):237-41

Institute of Human Genetics, Newcastle University, Newcastle upon Tyne NE1 3BZ, UK.

Nuclear RNA processing is a critical stage in eukaryotic gene expression, and is controlled in part by the expression and concentration of nuclear RNA-binding proteins. Different nuclear RNA-binding proteins are differentially expressed in different cells, helping the spliceosome to decode pre-mRNAs into alternatively spliced mRNAs. Recent post-genomic technology has exposed the complexity of nuclear RNA processing, and is starting to reveal the mechanisms and rules through which networks of RNA-binding proteins can regulate multiple parallel pathways. Identification of multiple parallel processing pathways regulated by nuclear RNA-binding proteins is leading to a systems-wide understanding of the rules and consequences of alternative nuclear RNA processing.
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http://dx.doi.org/10.1042/BST0380237DOI Listing
February 2010

Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-{beta}.

J Cell Sci 2010 Jan;123(Pt 1):40-50

IGBMC Department of Functional Genomics, Illkirch, France.

RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog beta (Tra2-beta), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-beta. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-beta repressed this exon. We propose that hRBMY associates with regions of the nucleus enriched in nascent RNA and participates in the regulation of specific splicing events in the germline by modulating the activity of constitutively expressed splicing factors.
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http://dx.doi.org/10.1242/jcs.055889DOI Listing
January 2010

The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon.

PLoS Genet 2009 Nov 6;5(11):e1000707. Epub 2009 Nov 6.

Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom.

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2beta is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5' splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2beta and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3' splice site normally weakly selected in the testis. Co-expression of Tra2beta with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.
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http://dx.doi.org/10.1371/journal.pgen.1000707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762042PMC
November 2009

Computational identification of 69 retroposons in Arabidopsis.

Plant Physiol 2005 Jun 27;138(2):935-48. Epub 2005 May 27.

National Center for Gene Research , Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China.

Retroposition is a shot-gun strategy of the genome to achieve evolutionary diversities by mixing and matching coding sequences with novel regulatory elements. We have identified 69 retroposons in the Arabidopsis (Arabidopsis thaliana) genome by a computational approach. Most of them were derivatives of mature mRNAs, and 20 genes contained relics of the reverse transcription process, such as truncations, deletions, and extra sequence additions. Of them, 22 are processed pseudogenes, and 52 genes are likely to be actively transcribed, especially in tissues from apical meristems (roots and flowers). Functional compositions of these retroposon parental genes imply that not the mRNA itself but its expression in gamete cells defines a suitable template for retroposition. The presence/absence patterns of retroposons can be used as cladistic markers for biogeographic research. Effects of human and the Mediterranean Pleistocene refugia in Arabidopsis biogeographic distributions were revealed based on two recent retroposons (At1g61410 and At5g52090). An evolutionary rate of new gene creation by retroposition was calculated as 0.6 genes per million years. Retroposons can also be used as molecular fossils of the parental gene expressions in ancient time. Extensions of 3' untranslated regions for those expressed parental genes are revealed as a possible trend of plant transcriptome evolution. In addition, we reported the first plant functional chimeric gene that adapts to intercompartmental transport by capturing two additional exons after retroposition.
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http://dx.doi.org/10.1104/pp.105.060244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1150409PMC
June 2005

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap.

J Biotechnol 2004 Apr;109(3):217-26

National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China.

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.
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http://dx.doi.org/10.1016/j.jbiotec.2003.12.012DOI Listing
April 2004

Sequence and analysis of rice chromosome 4.

Nature 2002 Nov;420(6913):316-20

National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai 200233, China.

Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.
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http://dx.doi.org/10.1038/nature01183DOI Listing
November 2002

A fine physical map of the rice chromosome 4.

Genome Res 2002 May;12(5):817-23

National Center for Gene Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China.

As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of 11 contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies.
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http://dx.doi.org/10.1101/gr.48902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC186569PMC
May 2002