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.

The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L) response to repeated drought stresses

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

Roles of alternative splicing in infectious diseases: from hosts, pathogens to their interactions / 中华医学杂志(英文版)

Alternative splicing (AS) is an evolutionarily conserved mechanism that removes introns and ligates exons to generate mature messenger RNAs (mRNAs), extremely improving the richness of transcriptome and proteome. Both mammal hosts and pathogens require AS to maintain their life activities, and inherent physiological heterogeneity between mammals and pathogens makes them adopt different ways to perform AS. Mammals and fungi conduct a two-step transesterification reaction by spliceosomes to splice each individual mRNA (named cis -splicing). Parasites also use spliceosomes to splice, but this splicing can occur among different mRNAs (named trans -splicing). Bacteria and viruses directly hijack the host's splicing machinery to accomplish this process. Infection-related changes are reflected in the spliceosome behaviors and the characteristics of various splicing regulators (abundance, modification, distribution, movement speed, and conformation), which further radiate to alterations in the global splicing profiles. Genes with splicing changes are enriched in immune-, growth-, or metabolism-related pathways, highlighting approaches through which hosts crosstalk with pathogens. Based on these infection-specific regulators or AS events, several targeted agents have been developed to fight against pathogens. Here, we summarized recent findings in the field of infection-related splicing, including splicing mechanisms of pathogens and hosts, splicing regulation and aberrant AS events, as well as emerging targeted drugs. We aimed to systemically decode host-pathogen interactions from a perspective of splicing. We further discussed the current strategies of drug development, detection methods, analysis algorithms, and database construction, facilitating the annotation of infection-related splicing and the integration of AS with disease phenotype.

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes / 南方医科大学学报

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Triple-negative breast cancer cells respond to T cells severely at the alternative splicing layer

BACKGROUND: Cross talk of tumor­immune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumor­immune microenvironment. RESULTS: Here, we re-exploited one transcriptomic dataset to gain insight into tumor­immune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factor­AS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. CONCLUSIONS: Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy.

Alternative Splicing Analysis of LACTB Gene and Expression Characteristics of Different Transcripts in Leukemia Cell Lines / 中国实验血液学杂志

OBJECTIVE@#To detect the expression of different transcripts of lactamase β（LACTB) gene in leukemic cell lines.@*METHODS@#NCBI website and DNAstar software were used to detect the Bioinformatics analysis of LACTB. The expression of different transcripts of LACTB gene in leukemic cell lines (THP-1, HL60, K562, U937, Jurkat and Raji) was detected by reverse transcription PCR (RT-PCR), DNA and clone sequencing; the expression of different transcripts of LACTB gene in leukemic cell lines was detected by Quantitative Real-time PCR.@*RESULTS@#There were a variety of splicing isomers in LACTB, and it could produce a variety of protein isomers with conserved N-terminal and different C-terminal, moreover, there were many splice isoforms of LACTB in leukemia cell lines, and there were different expression patterns in different cell lines, including XR1, V1, V2 and V3. The expression of total LACTB showed high in HL60 cells, while low in Raji cells, and the difference was statistically significant (P<0.05). The V1 was high expression in U937 cells but low in Raji cells, and the difference was statistically significant (P<0.05). V2 was high expression in HL60 cells but lowly in Raji cells, and the difference was statistically significant (P<0.05). The expression of V3 was low in THP-1 cells, which was significantly different as compared with that in normal bone marrow (P<0.05).@*CONCLUSION@#The reaserch found that there are many splice isomers of LACTB in leukemic cell lines, and there are different expression patterns in different cell lines.

Regulation effect of lipopolysaccharide on the alternative splicing and function of sweet taste receptor T1R2 / 华西口腔医学杂志

OBJECTIVES@#To identify the alternative splicing isoform of mouse sweet taste receptor T1R2, and investigate the effect of lipopolysaccharide (LPS) local injection on T1R2 alternative splicing and the function of sweet taste receptor as one of the bacterial virulence factors.@*METHODS@#After mouse taste bud tissue isolation was conducted, RNA extraction and reverse transcription polymerase chain reaction (PCR) were performed to identify the splicing isoform of T1R2. Heterologous expression experiments @*RESULTS@#T1R2 splicing isoform T1R2_Δe3p formed sweet taste receptors with T1R3, which could not be activated by sweet taste stimuli and significantly downregulated the function of canonical T1R2/T1R3. Local LPS injection significantly increased the expression ratio of T1R2_Δe3p in mouse taste buds.@*CONCLUSIONS@#LPS stimulation affects the alternative splicing of mouse sweet taste receptor T1R2 and significantly upregulates the expression of non-functional isoform T1R2_Δe3p, suggesting that T1R2 alternative splicing regulation may be one of the mechanisms by which microbial infection affects host taste perception.

Mechanisms of alternative splicing in regulating plant flowering: a review / 生物工程学报

Flowering is a critical transitional stage during plant growth and development, and is closely related to seed production and crop yield. The flowering transition is regulated by complex genetic networks, whereas many flowering-related genes generate multiple transcripts through alternative splicing to regulate flowering time. This paper summarizes the molecular mechanisms of alternative splicing in regulating plant flowering from several perspectives, future research directions are also envisioned.

Identification of mutations in the pah gene in pku patients in the state of mato grosso / Identificação de mutações no gene da pah em pacientes com fenilcetonúria do estado de mato grosso

ABSTRACT Objective: To identify phenylalanine hydroxylase (PAH) mutations in patients with phenylketonuria (PKU) from the Newborn Screening Service in Mato Grosso, Midwest Brazil. Methods: This is a cross-sectional descriptive study. The sample consisted of 19 PKU patients diagnosed by newborn screening. Molecular analysis: DNA extraction using the "salting-out" method. Detection of IVS10nt-11G>A, V388M, R261Q, R261X, R252W, and R408W mutations by the restriction fragment length polymorphism (RFLP) technique. Results: Two mutant alleles were identified in four patients (21.1%), one allele in five patients (26.2%), and none in the remaining ten patients (52.6%). A total of 13/38 alleles were detected, corresponding to 34.2% of the PAH alleles present. The most prevalent variant was V388M (13.2% of the alleles), followed by R261Q (10.1%) and IVS10nt-11G>A (7.9%). Three variants (R261X, R252W, and R408W) were not found. The most frequent mutation types were: missense mutation in eight alleles (18.4%) and splicing in four alleles (10.5%). The model proposed by Guldberg to determine a genotype/phenotype correlation was applied to four classical PKU patients with two identified mutations. In three of them, the predicted moderate/moderate or moderate PKU phenotype did not coincide with the actual diagnosis. The prediction coincided with the diagnosis of one classic PKU patient. The estimated incidence of PKU for Mato Grosso, Brazil, was 1:33,342 live births from 2003 to 2015. Conclusion: The only mutations found in the analyzed samples were the IVS10nt-11G>A, V388M, and R261Q. The genotype/phenotype correlation only occurred in four (5.3%) patients.

RESUMO Objetivo: Identificar mutações da fenilalanina hidroxilase (PAH) em pacientes com PKU (fenilcetonúria) do Serviço de Triagem Neonatal em Mato Grosso. Métodos: Estudo de corte transversal. Amostra composta de 19 pacientes com PKU através do exame de triagem neonatal biológica. Análise molecular: a) extração de DNA pela metodologia "salting out". B) detecção de mutações IVS10nt-11G>A, V388M, R261Q, R261X, R252W e R408W pela técnica de polimorfismo de comprimento de fragmento de restrição (RFLP). Resultados: Dois alelos foram identificados em quatro pacientes (21,1%), um alelo em cinco pacientes (26,2%) e nenhum nos dez pacientes restantes (52,6%). Um total de 13/38 alelos foram identificados, correspondendo a 34,2% dos alelos PAH presentes. A variante mais prevalente foi a V388M (13,2% dos alelos), seguida de R261Q (10,1%) e IVS10nt-11G>A (7,9%). Três variantes (R261X, R252W e R408W) não foram encontradas. Os tipos de mutações mais frequentes foram: troca de sentido em oito alelos (18,4%) e emenda em quatro alelos (10,5%). O modelo proposto por Guldberg para determinar uma correlação genótipo/fenótipo foi aplicado para quatro pacientes clássicos de PKU, com duas mutações identificadas. Em três, o fenótipo previsto de PKU moderada/moderada ou moderada não coincidiu com o diagnóstico real. A predição coincidiu com o diagnóstico de um paciente PKU clássico. A incidência de PKU estimada para Mato Grosso, Brasil foi de 1:33.342 nascidos vivos para o período de 2003 a 2015. Conclusões: Foram encontradas apenas as mutações IVS10nt-11G>A, V388M, R261Q nas amostras analisadas. A correlação genótipo/fenótipo ocorreu em quatro (5,3%) pacientes.

Bioinformatics Analysis and Preliminary Functional Study of Abnormal Expression of Splicing Factors in Gastric Cancer / 中国医学科学院学报

To analyze the expression of splicing factors in gastric cancer using bioinformatics methods and investigate the effect of aberrantly expressed serine/arginine-rich splicing factor(SRSF10)on the phenotype of gastric cancer cells. The RNA-seq data of gastric cancer and paracancerous tissues were downloaded from The Cancer Genome Atlas(TCGA)cancer database,and bioinformatics analysis was performed to obtain the splicing factors differentially expressed in gastric cancer.The splicing factor SRSF10 was selected to investigate its effect on the development of gastric cancer.RNA interference technology was used to construct SRSF10 knockdown gastric cancer cells.MTS,Transwell,and cell scratches were used to study the effect of SRSF10 knockdown on gastric cancer cell phenotype. A total of 48 splicing factors were identified in gastric cancer by a series of bioinformatics techniques,of which 35 were up-regulated and 13 were down-regulated.The splicing factor SRSF10,which was up-regulated,was selected for further study.It was found that the gastric cancer cells after SRSF10 knockdown proliferated more slowly and had lower migration ability than normal gastric cancer cells. Multiple splicing factors are found in gastric cancer and may play an important role in the development of gastric cancer.The splicing factor SRSF10 may contribute to the pathogenesis of gastric cancer.

mTOR-coordinated Post-Transcriptional Gene Regulations: from Fundamental to Pathogenic Insights

Post-transcriptional regulations of mRNA transcripts such as alternative splicing and alternative polyadenylation can affect the expression of genes without changing the transcript levels. Recent studies have demonstrated that these post-transcriptional events can have significant physiological impacts on various biological systems and play important roles in the pathogenesis of a number of diseases, including cancers. Nevertheless, how cellular signaling pathways control these post-transcriptional processes in cells are not very well explored in the field yet. The mammalian target of rapamycin complex 1 (mTORC1) pathway plays a key role in sensing cellular nutrient and energy status and regulating the proliferation and growth of cells by controlling various anabolic and catabolic processes. Dysregulation of mTORC1 pathway can tip the metabolic balance of cells and is associated with a number of pathological conditions, including various types of cancers, diabetes, and cardiovascular diseases. Numerous reports have shown that mTORC1 controls its downstream pathways through translational and/or transcriptional regulation of the expression of key downstream effectors. And, recent studies have also shown that mTORC1 can control downstream pathways via post-transcriptional regulations. In this review, we will discuss the roles of post-transcriptional processes in gene expression regulations and how mTORC1-mediated post-transcriptional regulations contribute to cellular physiological changes. We highlight post-transcriptional regulation as an additional layer of gene expression control by mTORC1 to steer cellular biology. These emphasize the importance of studying post-transcriptional events in transcriptome datasets for gaining a fuller understanding of gene expression regulations in the biological systems of interest.

Synonymous variants of the ATP7B gene may cause abnormal splicing of mRNA by affecting the exonic splicing enhancers / 中华医学遗传学杂志

OBJECTIVE@#To explore the effect of rare synonymous variants of the ATP7B gene on the splicing of its precursor mRNA.@*METHODS@#A total of 248 rare synonymous variants with allelic frequency of T (p.L540L) and c.3888C>T (p.A1296A) variants could lead to abnormal splicing of the corresponding exons, resulting in complete skipping of exon 4 and 25% increase in the skipping of exon 18, respectively.@*CONCLUSION@#Synonymous variants may affect the splicing of precursor mRNA in various ways, particularly the destruction of ESE motif. This study confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants can affect the mRNA splicing of the ATP7B gene, resulting in skipping of corresponding exons, which may provide a basis for genetic diagnosis and consultation of carriers.

Scavenger Receptor Class A to E Involved in Various Cancers / 전남의대학술지

Scavenger receptors typically bind to multiple ligands on a cell surface, including endogenous and modified host-derived molecules and microbial pathogens. They promote the elimination of degraded or harmful substances such as non-self or altered-self targets through endocytosis, phagocytosis, and adhesion. Currently, scavenger receptors are subdivided into eight classes based on several variations in their sequences due to alternative splicing. Since recent studies indicate targeting scavenger receptors has been involved in cancer prognosis and carcinogenesis, we will focus on the current knowledge about the emerging role of scavenger receptor classes A to E in cancer progression.

Genetic study of a family of neuronal ceroid lipofuscinosis caused by a heterozygous mutation of gene / 浙江大学学报·医学版

OBJECTIVE@#To analyze the genetic cause of a family with autosomal recessive neuronal ceroid lipofuscinoses (NCL).@*METHODS@#The proband was screened for mutations within the coding region of the candidate genes through high-throughput targeted sequencing. Potential causative mutations were verified by PCR and Sanger sequencing in the proband and his parents. RT-PCR and TA clone sequencing were performed to investigate whether the mRNAs were abnormally spliced.@*RESULTS@#The sequencing results revealed compound heterozygous mutations of :c.486+2T>C and c.486+4A>T, which were respectively inherited from his parents. RT-PCR and TA cloning sequencing suggested that the mRNAs were abnormally spliced in two forms due to both mutations.@*CONCLUSIONS@#The compound heterozygous mutations of :c.486+2T>C and c.486+4A>T are possibly the genetic causes of the NCL family. Detection of the novel mutation has extended mutation spectrum of .

Identification of neoantigens derived from alternative splicing and RNA modification

The acquisition of somatic mutations is the most common event in cancer. Neoantigens expressed from genes with mutations acquired during carcinogenesis can be tumor-specific. Since the immune system recognizes tumor-specific peptides, they are potential targets for personalized neoantigen-based immunotherapy. However, the discovery of druggable neoantigens remains challenging, suggesting that a deeper understanding of the mechanism of neoantigen generation and better strategies to identify them will be required to realize the promise of neoantigen-based immunotherapy. Alternative splicing and RNA editing events are emerging mechanisms leading to neoantigen production. In this review, we outline recent work involving the large-scale screening of neoantigens produced by alternative splicing and RNA editing. We also describe strategies to predict and validate neoantigens from RNA sequencing data.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Análise Comparativa da Diversidade de Splicing Alternativo no Proteoma do Cérebro Humano e Murino / Comparative Analysis of the Diversity of Alternative Splicing in the Human and Murine Brain Proteome

Os avanços obtidos em transcriptômica, em função do desenvolvimento de sequenciadores de alta vazão, e na proteômica, por meio dos modernos espectrômetros de massas (MS), resultaram em um grande volume de dados que passou a ser integrado em diversos estudos de Bioinformática, levando ao melhor entendimento sobre a fração dos RNAs mensageiros efetivamente traduzida em proteínas. A proteogenômica é a área de pesquisa que reúne estas tecnologias, atuando na interface entre a genômica e a proteômica para interpretar eventos moleculares, tais como, por exemplo, o splicing alternativo. Este evento molecular é capaz de gerar RNAs mensageiros diferentes a partir de um mesmo gene, podendo alterar a sequência polipeptídica e, consequentemente, gerar proteoformas com funções distintas. Neste sentido, atualmente alguns projetos têm realizado esta análise integrativa com o intuito de comparar os resultados de amostras de ser humano e outros mamíferos, uma vez que alguns destes animais são utilizados como organismos modelo para o estudo dos aspectos moleculares de doenças, como as neurodegenerativas. Desta forma, este projeto teve como objetivo principal analisar o perfil de expressão de variantes de splicing alternativo em dados de espectrometria de massas de proteínas de amostras de tecidos de cérebros sadios de humano e camundongo

Para tal, foram utilizados dados de mRNAs de referência (Refseq), ESTs e sequências da base de dados Uniprot/Swiss-Prot para confecção de repositórios de sequências proteicas personalizados, utilizando uma metodologia desenvolvida pelo nosso grupo de pesquisa denominada matrizes ternárias. O repositório personalizado para humano continha 20.150 sequências canônicas e 204.294 peptídeos não redundantes, totalizando 224.453 sequências. O repositório de camundongo possuía 16.888 sequências canônicas e 156.889 peptídeos não redundantes, totalizando 173.777 sequências. Estes repositórios de sequências proteicas personalizados foram empregados para a análise de dados de espectrometria de massas de três regiões distintas do cérebro de humano e camundongo (corpo caloso, nervo óptico e bulbo olfatório). A partir destas análises, nós inferimos a expressão de um total de 3.289 proteínas canônicas e 23 proteoformas de genes ortólogos entre humano e camundongo. Dentre as proteoformas, seis foram inferidas a partir de peptídeos proteotípicos idênticos identificados em dados de MS de humano e camundongo (PKM, CRMP1, PRKCB, STXBP1, CADM1 e HNRNPK). Portanto, acreditamos que a identificação de peptídeos compartilhados entre humano e camundongo, pertencentes a proteoformas de genes ortólogos, realizada neste projeto, contribuiu para o melhor conhecimento da diversidade de splicing do cérebro de humano e camundongo. (AU)

Increasing evidence for the presence of alternative proteins in human tissues and cell lines

Recent findings coming from human proteome research employing mass-spectrometry and ribosomal profiling methods have provided evidence for the translation of non-annotated coding sequence (CDSs) into alternative proteins (APs). The presence of APs in many human tissues and cell lines may become an important issue in genome sciences, especially in cancer genomics where the frequency of alternative proteins seems to be 10-fold higher than normal tissues. Finding new proteins can impact medical research by filling gaps in known molecular pathways or revealing new molecular markers and therapeutic targets. Among the cellular processes possibly involved in protein diversity, alternative splicing (AS) is the most cited, and it consists of an often-regulated mechanism that generates different mRNAs from the same gene, contributing to the functional diversity of mammalian cells. In the past, evidence for AS from multi-exon genes have come mainly from expression sequence tag (EST) data; only recently has mass-spectrometry (MS) been used to investigate the translation of alternative transcripts. Exploration of human MS data has detected tens to hundreds of alternative proteins in normal tissues, and thousands in cancer cell lines, suggesting that alternative proteins may have an important role in cancer. Analysis of MS data has revealed a vastly diverse AP repertoire, with some of this diversity being exclusively detected in cancer cells. Proteomic characterization of 20 breast cancer cell lines revealed a surprising 1,860 protein variants resulting from AS. Among these, 4 AP are clearly involved in cancer. A truncated variant of the NF- kB p65 subunit, a truncated form of the focal adhesion kinase PTK2 and two CD47 transmembrane receptor protein variants. Until now, little is known about the functional differences between these variants. Another cellular mechanism that possibly creates protein diversity is the alternative usage of translation initiation site (TIS). Detection of TIS is made possible by the Ribosome Profiling (RP) method. The principle of this technique is to capture mRNA translation by freezing the actively translating ribosomes onto transcripts, and then separating them by ultracentrifugation. Recently, RP was applied to mouse embryonic fibroblast cells and human HEK293 cells. The results revealed that the majority of mRNAs contain more than one translation initiation site (TIS), with more than 50% of the detected TISs mapping to alternative ORFs. In this review, we present a list of human alternative proteins validated by small and large-scale experimental methods. We also highlight that APs are probably not a secondary product of inaccurate splicing or translational process and most likely play an important role in the tumorigenic process. Thus, APs constitutes a promising research line for basic and clinical aspects of cancer (AU)

Analysis of ATP2A2 gene mutations in a pedigree and a sporadic case with Darier disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect mutations of ATP2A2 gene in a pedigree and a sporadic case with Darier disease (DD) and explore the underlying molecular mechanism.</p><p><b>METHODS</b>Clinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from blood samples of four members from the pedigree (including three patients and one healthy member), the sporadic case and 100 healthy controls. PCR was performed to amplify all coding exons of the ATP2A2 gene. And the products were directly sequenced to detect mutations.</p><p><b>RESULTS</b>A missense mutation c.1484C>T (p.S495L) in exon 12 was detected in all patients of the pedigree. For the sporadic case, a novel splicing mutation c.325-2A>G was detected at the junction between intron 4 and exon 5. The same mutations were not found in the 100 healthy controls.</p><p><b>CONCLUSION</b>Mutations of the ATP2A2 gene may lead to the occurrence of DD in both familial and sporadic cases with DD.</p>

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing / 결핵및호흡기질환

BACKGROUND: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. METHODS: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RESULTS: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. CONCLUSION: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.