Investigation of androgen receptor-dependent alternative splicing has identified a unique subtype of lethal prostate cancer / 亚洲男科学杂志(英文版)

A complete proteomics study characterizing active androgen receptor (AR) complexes in prostate cancer (PCa) cells identified a diversity of protein interactors with tumorigenic annotations, including known RNA splicing factors. Thus, we chose to further investigate the functional role of AR-mediated alternative RNA splicing in PCa disease progression. We selected two AR-interacting RNA splicing factors, Src associated in mitosis of 68 kDa (SAM68) and DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5) to examine their associative roles in AR-dependent alternative RNA splicing. To assess the true physiological role of AR in alternative RNA splicing, we assessed splicing profiles of LNCaP PCa cells using exon microarrays and correlated the results to PCa clinical datasets. As a result, we were able to highlight alternative splicing events of clinical significance. Initial use of exon-mini gene cassettes illustrated hormone-dependent AR-mediated exon-inclusion splicing events with SAM68 or exon-exclusion splicing events with DDX5 overexpression. The physiological significance in PCa was investigated through the application of clinical exon array analysis, where we identified exon-gene sets that were able to delineate aggressive disease progression profiles and predict patient disease-free outcomes independently of pathological clinical criteria. Using a clinical dataset with patients categorized as prostate cancer-specific death (PCSD), these exon gene sets further identified a select group of patients with extremely poor disease-free outcomes. Overall, these results strongly suggest a nonclassical role of AR in mediating robust alternative RNA splicing in PCa. Moreover, AR-mediated alternative spicing contributes to aggressive PCa progression, where we identified a new subtype of lethal PCa defined by AR-dependent alternative splicing.

Splicing abnormalities caused by a novel mutation in the PHKA2 gene in children with glycogen storage disease type IX / 中华肝脏病杂志

Objective: Glycogen storage disease type IX (GSD-IX) is a rare primary glucose metabolism abnormality caused by phosphorylase kinase deficiency and a series of pathogenic gene mutations. The clinical characteristics, gene analysis, and functional verification of a mutation in a child with hepatomegaly are summarized here to clarify the pathogenic cause of the disease. Methods: The clinical data of a child with GSD-IX was collected. Peripheral blood from the child and his parents was collected for genomic DNA extraction. The patient's gene diagnosis was performed by second-generation sequencing. The suspected mutations were verified by Sanger sequencing and bioinformatics analysis. The suspected splicing mutations were verified in vivo by RT-PCR and first-generation sequencing. Results: Hepatomegaly, transaminitis, and hypertriglyceridemia were present in children. Liver biopsy pathological examination results indicated glycogen storage disease. Gene sequencing revealed that the child had a c.285 + 2_285 + 5delTAGG hemizygous mutation in the PHKA2 gene. Sanger sequencing verification showed that the mother of the child was heterozygous and the father of the child was of the wild type. Software such as HSF3.1 and ESEfinder predicted that the gene mutation affected splicing. RT-PCR of peripheral blood from children and his mother confirmed that the mutation had caused the skipping of exon 3 during the constitutive splicing of the PHKA2 gene. Conclusion: The hemizygous mutation in the PHKA2 gene (c.285 + 2_285 + 5delTAGG) is the pathogenic cause of the patient's disease. The detection of the novel mutation site enriches the mutation spectrum of the PHKA2 gene and serves as a basis for the family's genetic counseling.

The Mechanisms of the Production of Different Isoforms of Nuclear Factor E2-related Factor 1 and Their Role in Diseases / 中国生物化学与分子生物学报

During the normal process of biological growth and development, NF-E2-related factor 1 (Nrf1/ Nfe2l1) plays a unique role in maintaining intracellular homeostasis and organ integrity. The deficiency of Nrf1 will lead to severe oxidative stress, genomic instability, and result in liver cancer, neurodegenerative disorder and other diseases. In recent years, it has been found that Nrf1 could yield different activated isoforms or even opposite activity isoforms in a variety of ways to perform distinct functions, and the distribution of these isoforms may play a vital role in the process of tumor development. Therefore, to gain a better understanding of the function of Nrf1 isoforms in cells and tissues, we first briefly introduced its discovery process, and demonstrated the multiple mechanisms of distinct isoform production including selective shear processing, internal selective translation initiation, and post-translation shear processing. More importantly, three different post-translational processing models, transmembrane dynamic processing, site-specific processing, ubiquitin-dependent processing, were expounded in detail. Furthermore, the biological function of different isoforms of Nrf1 and its role in diseases were also summarized in the last section. Collectively, we focus on the production mechanism of different isoforms of Nrf1 and their roles in diseases so as to lay a foundation for finding new strategies for tumor treatment.

Power-law Behavior of the Alternative Splicing of Exons in Human Transcriptome

Aims: To establish the common rules of exon combinatorics during RNA splicing. Study Design: Inferring a plausible statistical model of exon combinatorics from the annotated models of human genes during RNA splicing. Place and Duration of Study: Department of Genetics (Belarusian State University), Proteome and Genome Research Unit (Luxembourg Institute of Health), Department of Genetics (Lomonosov Moscow State University) and Moscow Center of Experimental Embryology and Reproductive Biotechnologies, between January 2017 and July 2019. Methodology: We used human mRNA and EST sequences from GenBank (1093522 unique records in total) and linear models of the human genes from Ensembl (58051 genes), AceView (72384 genes), ECgene (57172 genes), NCBI RefSeq (54262 genes), UCSC Genome Browser (58037 genes) and VEGA (54950 genes) to calculate a combinatorial index of human exons. We inferred the most plausible statistical model describing the distribution of combinatorial index of human exons using Clauset’s mathematical formalism. Predictors of the combinatorial index values and functional outcomes of the predefined behavior of exons during splicing were also determined. Results: Power-law is the most plausible statistical model describing the combinatorics of exons during RNA splicing. The combinatorial index of human exons is defined by more than 90% by the 138 features that have different importance. The most important of these features are the abundance of exon in transcripts, the strength of splice sites, the rank of exon in transcripts and the type of exon. Analysis of the marginal effects shows that different values of the same feature have unequal influence on the combinatorial index of human exons. Power-law behavior of exons during RNA splicing pre-determines structural diversity of transcripts, low sensitivity of splicing process to random perturbations and its high vulnerability to manipulation with highly combinative exons. Conclusion: Exons widely involved in alternative splicing are a part of the common power-law phenomenon in human cells. The power-law behavior of exons during RNA splicing gives the unique characteristics to human genes.

Hepatitis B spliced protein enhanced NF-κB activities via interaction with ubiquitously expressed transcript splice variant 1 / 中国人兽共患病学报

We investigated whether hepatitis B spliced protein affect the NF-kappa B activities by interacting with ubiquitously expressed transcript splice variant 1 (UXT-V1).The HBSP-UXT-V1 protein interactions were screened by yeast two hybrid assay,and confirmed by immunoprecipitation,confocal microscopy,mammalian two hybrid assay,and GST-Pulldown assay.The reporter plasmids driven by NF-kappa B promoter were transfected into UXT-knockdown HBSP stably expressed cell lines,and the reporter genes were detected after transfection.Results showed that the interaction between UXT-V1 and HBSP in yeast was demonstrated.Furtherly,HBSP could interact with UXT-V1 in mammalian cells.HBSP could enhance NF-kappa B activities,and this effect was partly achieved by the interaction with UXT-V1.In conclusion,the effect of HBSPUXT-V1 interaction on the NF-kappa B pathway in hepatocytes may have an impact on HBV related liver diseases.

Hepatitis B spliced protein triggers TGFβ1-induced epithelial-mesenchymal transition via interaction with transforming growth factor beta-1-induced transcript 1 protein / 中国人兽共患病学报

To investigate the TGFβ1-induced epithelial-mesenchymal transition (EMT) of Huh7 hepatoma cells caused by interaction of hepatitis B spliced protein (HBSP) with transforming growth factor beta-1-induced transcript 1 protein (TGFβ31I1),coding region of HBSP was cloned into lentiviral expression vector.Huh7 hepatoma cells were infected by recombinant lentivirus packaged in 293T cells.Stable cell lines expressing HBSP or control cells were selected by puromycin.Cells were incubated with 5 ng/mL TGFβ1 for 24 h,and observed under contrast-phase microspcope.Then the whole cell lysates were collected for western blot analysis using specific antibodies against EMT markers including E-cadherin,N-cadherin,Claudin-1 and β-catenin.To evaluate the effects of HBSP-TGFβ1I1 interaction on EMT,TGFβ1-induced EMT marker transition,as well as cell invasion and migration were explored after knocking down of TGFβ1I1 by siRNA.Results showed that Huh7 cell lines expressing HBSP (Huh7-HBSP flag-HIV) and control cell lines (Huh7-flag-HIV) were successfully established.Huh7-HBSP flag-HIV cells lost their pebble-like shape and tight cell-cell adhesion and transformed into the mesenchymal-like cells in the presence of TGFβ1.Decreased expression level of epithelial marker of E-cadherin,Claudin-1,β-catenin,increased expression level of mesenchymal marker of N-cadherin,and enhanced migration and invasion abilities were observed in Huh7-HBSP-flag-HIV cells as compared to the control cells.Moreover,the changes of EMT markers and metastasis abilities of Huh7-HBSP-flag-HIV cells could be reversed when TGFβ111 was knocked down by siRNA.In conclusion,HBSP could promote hepatoma cell migration and invasion by triggering EMT via interaction with TGFβ111.Our findings highlight new insights for HBSP-induced HCC progression.

Inhibición parcial de dos genes que codifican para proteínas del empalmosoma en Giardia intestinalis / Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis

Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.

Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

Plaquetas: papéis tradicionais e não tradicionais na hemostasia, na inflamação e no câncer / Platelets: traditional and nontraditional roles in hemostasis, inflammation and cancer

A principal e mais conhecida função plaquetária ainda está relacionada à parada de sangramento após um dano vascular. No entanto, plaquetas estão envolvidas em diversos processos, tais como iniciar e amplificar a inflamação, interagir com células da resposta imune, além de participar na progressão tumoral, angiogênese e metástase. Neste sentido, está claro que plaquetas apresentam funções no processo inflamatório e podem influenciar respostas imune, além de desordens plaquetárias autoimune erelacionadas a presença de auto-anticorpos após transfusões, comopor exemplo, na lesão pulmonar aguda associada à transfusão. Após muita especulação, recentes observações têm estabelecido novos paradigmas relacionando plaquetas à biologia molecular. Plaquetas humanas contêm fatores de spliceossomo, incluindo pequenos RNAs nucleares, proteínas de splicing e pre-mRNA endógenos. Outro ponto importante é o controle do número de plaquetas circulantes, resultado do equilíbrio entre a produção e destruição dessas células. Assim, é proposto um processo de morte programada da célula anucleada que determina seu tempo de vida. Esse processo é alvo de especulações desde a década de 60 e ainda permanece em discussão. A noção geral de que plaquetas funcionais são importantes para o sucesso de processos hematogênicos corroboram com inovações experimentais e também ligam a processos de interação plaquetas-células tumorais e seu microambiente que regula a progressão maligna. Plaquetas contribuem na sobrevivência e disseminação de células tumorais. Desta forma, discutimos aqui os mecanismos pelos quais as plaquetas atuam na imunidade, na inflamação e no câncer, uma vez que estas pequenas células são mais versáteis do que se pensava.

The principal and the most known function of platelets still remains stopping hemorrhage following vascular injury. However, platelets are involved in diverse processes such as triggering inflammation, participating in the immune response, besides tumor progression, angiogenesis, and metastasis. In this sense, it is becoming increasingly clear that platelets display inflammatory functions and can influence both innate and adaptive immune responses, such as autoimmune and alloimmune platelet disorders, and transfusion-related acute lung injury (TRALI). Despite much speculation recent observations have established new paradigms relevant to influence of platelets on molecular biology. Primary human platelets contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. Other point is, like all lineages of blood cells, the steady state number of mature platelets is the result of a balance between their production and destruction. Thus, it isproposed a programmed anuclear cell death delimits platelet lifespan is subject of speculation since the 1960s and has remainedelusive. The general notion that functional platelets are importantfor successful hematogenous tumor metastasis dates more than 4 decades and has been corroborated in numerous experimentalsettings. The dynamic crosstalk between tumors and their microenvironment is increasingly recognized as a key regulator ofmalignant progression. These contributions of platelets to tumorcell survival and spread suggest platelets as a new avenue forresearch. Here, we discuss the mechanisms by which plateletscontribute to immunity, inflammation, and cancer, since thesesmall cells are more versatile than we once thought.

Identification of a Novel Intronic Transcript Containing 11q13.5 HERV-W gag Sequence / 天津医药

Objective:To identify the novel intronic transcripts containing 11q13.5 HERV-W gag sequence, and explore the modulation of the transcripts on the alternative splicing of host gene PTD015. Methods:Half-nested PCR and touchdown PCR were used to amplify the target transcripts. The transcripts were cloned and sequenced. The plasmids inserted with the target transcripts were transfected into JEG3 cells and the alternative spliced mRNA levels of PTD015 were measured with real time PCR. Results:A 1 739 bp novel intronic transcript containing 755 bp 11q13.5 HERV-W gag sequence, 527 bp 5′ long terminal repeat and 457 bp sequence on the 5′-end of 11q13.5 HERV-W was identified. The intronic antisense transcripts significantly down-regulated the alternative spliced mRNA levels of PTD015. Conclusion:The intronic antisense transcripts originating from the second intron of gene PTD015 could modulate the alternative splicing of the host gene PTD015.

Yeast two-hybrid system screening liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome / 中华微生物学和免疫学杂志

Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activatian capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatacytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AHI09 yeast cells. Four liver cell proteins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.

Effects of the splicing-specific protein on the regulatory elements of hepatitis B virus per se / 中华微生物学和免疫学杂志

Objective To investigate the effects of the novel protein,TPds encoded by the 2.2 kb doubly spliced variant of hepatitis B virus(HBV)genome on the regulatory elements of HBV.Methods HBV promoters and enhancers were amplified by PCR and respectively cloned into the plasmid pGL3-basic to construct the recombinant firefly luciferase reporter vectors including pGL3-BCP(harboring basal core promoter),pGL3-CP1601(core promoter with enhancer Ⅱ),pGL3-XP(minimal X gene promoter),pGL3 XP1071(X gene promoter with enhancer Ⅰ),pGL3-SP1(promoter of large surface antigen)and pGL3-SP2 (promoter of middle surface antigen).The recombinant reporter plasmids were respectively co-transfected with either the pcDNA3.1/HisC-TPds(TPd8 expression plasmid)or pcDNA3.1/HisC(empty control)into Huh7 hepatocytes by FuGENE6 transfection reagent.Cells were lysed 48 h post transfection,the intracellulax luciferase activities were monitored and calculated by SPSS11.5 software.Results As compared with the empty control of pcDNA3.1/HisC.the intracellular luciferase activities were elevated when the Huh7 hepatocytes were co-transfected by pcDNA3.1/HisC-TPds with one of the recombinant reporter plasmids as pGL3-CP1601,pGL3-XP1071,pGL3-SP1,or pGL3-SP2,while no changes with pGL3-BCP or pGL3-XP.Conduslon TPds could transactivate the HBV regulatory elements a8 promoter SP1,SP2.and enhancer Ⅰ,Ⅱ,while no influences on basal core promoter and minimal X gene promoter.

The anti-IFN-α effects of the novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virus genome / 中华微生物学和免疫学杂志

Objective To investigate the anti-IFN-α effects of the novel protein TSR'r' encodedby the 458 nt-1308 nt spliced variant of hepatitis B virus genome,and to determine its functional domaias.Methods the TSR'r' gene(originated from open reading frame of HBV DNA polymerase,T represents terminal protein region,S represents the Spacer region,R'represents the truncated reverse transcriptase region,and r'represents the truncated RNaseH region)of the 458 nt-1308 nt spliced variant of HBV genome and its deletants were amplified by PCR and were cloned into the pcDNA3.1/HisC vector.The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent,and the expression of the fusion protein was detected by Western blot.Huh7 hepatocytes were co-transfected with p6 16CAT and the recombinant vector harboring either TSR'r'or the related deletant,and treated with IFN-α 2a 48 h post transfection.After 24 h stimulating.the cells were lysed and the intracellular CAT value was calculated.All data were processed with One-way analysis of variance(ANOVA).Resuits Recombinant vectors harboring either the TSR'r'gene or related deletant were constructed successfully,and the fusion proteins were expressed well in Huh7 cells.When Huh7 hepatocytes were co-transfected with p6-16CAT and TSR'r' recombinant.the intracellular CAT values reduced gradually as paralleled with the increasing amount of TSR'r'recombinant.Furthermore,as compared with the empty vector,intracellular CAT values also decreased significantly when the Huh7 cells co-transfected with recombinant harboring TP plus Spacer regions,while any of the other deletants(harboring either TP or Spacer region or neither)showed no significant difference.Conclusion The novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virns genome suppressed the response of Huh7 hepatocytes to IFN-α.and the N-terminal TP plus Spacer region was the functional domain of the protein for anti-IFN-α effects.

Yeast Genetics Revisited / 生物化学与生物物理进展

With the development of genetics, more and more research has been focused on modal organisms because of the conservative nature of biological species. Saccharomyces cerevisiae is one of the most important model organisms widely used all over the world. Recently, works published in this journal from Tsinghua University revealed a role of YMR166C in mitochondrial magnesium homeostasis in Saccharomyces cerevisiae with transposon-based genetic screen. A genome-wide screening in Saccharomyces cerevisiae for suppressor genes of MTM1 also yielded some very interesting results. Those wonderful works indicated a new era in genetic studies in China.

Structural analysis of spliced variants of hepatitis B virus genomes / 中华传染病杂志

Objective To study the structure of spliced variants of hepatitis B virus (HBV) genomes and elucidate their potential pathogenicity. Methods Amplified the spliced variants of hepatitis B virus genomes by mean of PCR from the serum of the patients with chronic hepatitis B, sequenced and analysis the characteristics of such genomes. Results 10 different types of the spliced variants of hepatitis B virus genomes were obtained with the molecular weight ranging from 765 bp to 2039 bp. There were 6 splicing donor sites and 6 accepter sites, respectively in HBV genomes. All spliced variants showed one or more deletions in the regions coding for core, preS1, preS2 and surface protein, while retained thepathogenic X gene and cis elements which were essential for viral replication and packaging. Conclusions Spliced variants of hepatitis B virus genomes were commonly detected in the serum from chronic Hepatitis B patients, the characteristic structure of such variants implied that they might closely co-related with the pathogenicity of HBV.