Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription

Abstract\ud \ud \ud \ud Background\ud \ud RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression. However, the complement of human genes in which introns are transcribed, and the number of intronic transcriptional units and their tissue expression patterns are not known.\ud \ud \ud \ud Results\ud \ud A survey of mRNA and EST public databases revealed more than 55,000 totally intronic noncoding (TIN) RNAs transcribed from the introns of 74% of all unique RefSeq genes. Guided by this information, we designed an oligoarray platform containing sense and antisense probes for each of 7,135 randomly selected TIN transcripts plus the corresponding protein-coding genes. We identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney. The most highly expressed antisense TIN RNAs were transcribed from introns of protein-coding genes significantly enriched (p = 0.002 to 0.022) in the 'Regulation of transcription' Gene Ontology category. RNA polymerase II inhibition resulted in increased expression of a fraction of intronic RNAs in cell cultures, suggesting that other RNA polymerases may be involved in their biosynthesis. Members of a subset of intronic and protein-coding signatures transcribed from the same genomic loci have correlated expression patterns, suggesting that intronic RNAs regulate the abundance or the pattern of exon usage in protein-coding messages.\ud \ud \ud \ud Conclusion\ud \ud We have identified diverse intronic RNA expression patterns, pointing to distinct regulatory roles. This gene-oriented approach, using a combined intron-exon oligoarray, should permit further comparative analysis of intronic transcription under various physiological and pathological conditions, thus advancing current knowledge about the biological functions of these noncoding RNAs.The authors thank Camila Egidio for help with testing the Agilent microarray protocol. The authors also thank Dr Marcia Kubrusly (Hospital das Clínicas, Universidade de São Paulo) and Dr Marcello Barcinski (Instituto Nacional de Câncer, Rio de Janeiro) for providing the tissue samples. This work was supported by a grant from Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP to SVA, EMR and AMDS and by fellowships from FAPESP and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brasil.The authors thank Camila Egidio for help with testing the Agilent microarray protocol. The authors also thank Dr Marcia Kubrusly (Hospital das Clínicas, Universidade de São Paulo) and Dr Marcello Barcinski (Instituto Nacional de Câncer, Rio de Janeiro) for providing the tissue samples. This work was supported by a grant from Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP to SVA, EMR and AMDS and by fellowships from FAPESP and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brasil

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao PauloFAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)EMREMRCNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq, Brasi

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma is very common in head and neck cancer, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from 13 larynx tumors and 10 non-neoplastic larynx tissues using a custom-built cDNA microarray containing 331 probes for 284 genes previously identified by informatics analysis of EST databases as markers of head and neck tumors. Thirty-five genes showed statistically significant differences (SNR >= 11.01, p <= 0.001) in the expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction and transcriptional regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise non-annotated exons or as yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. The differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated with an antimetastatic function. Interestingly, CSTB showed a low expression in the tumor samples analyzed (p<0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and provide candidate markers for improving diagnosis, prognosis and treatment of this carcinoma.Fundacao de Amparo A Pesquisa do Estado de Sdo Paulo (FAPESP), BrazilFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ludwig Institute for Cancer ResearchLudwig Institute for Cancer Researc

Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations.

The biological role of extracellular vesicles (EVs) in diffuse large B-cell lymphoma (DLBCL) initiation and progression remains largely unknown. We characterized EVs secreted by 5 DLBCL cell lines, a primary DLBCL tumor, and a normal control B-cell sample, optimized their purification, and analyzed their content. We found that DLBCLs secreted large quantities of CD63, Alix, TSG101, and CD81 EVs, which can be extracted using an ultracentrifugation-based method and traced by their cell of origin surface markers. We also showed that tumor-derived EVs can be exchanged between lymphoma cells, normal tonsillar cells, and HK stromal cells. We then examined the content of EVs, focusing on isolation of high-quality total RNA. We sequenced the total RNA and analyzed the nature of RNA species, including coding and noncoding RNAs. We compared whole-cell and EV-derived RNA composition in benign and malignant B cells and discovered that transcripts from EVs were involved in many critical cellular functions. Finally, we performed mutational analysis and found that mutations detected in EVs exquisitely represented mutations in the cell of origin. These results enhance our understanding and enable future studies of the role that EVs may play in the pathogenesis of DLBCL, particularly with regards to the exchange of genomic information. Current findings open a new strategy for liquid biopsy approaches in disease monitoring

Expression signatures of antisense PIN RNAs and corresponding PIN RNA-overlapped exon pairs relative to their 3' protein-coding exons

<p><b>Copyright information:</b></p><p>Taken from "Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription"</p><p>http://genomebiology.com/2007/8/3/R43</p><p>Genome Biology 2007;8(3):R43-R43.</p><p>Published online 26 Mar 2007</p><p>PMCID:PMC1868932.</p><p></p> A subset of 64 pairs of antisense PIN RNAs and corresponding PIN RNA-overlapped exons were identified among the tissue signatures shown in Figure 10 as having correlated patterns of expression: 49 pairs were identified in which the 3' exon of the protein-coding transcript (right panel) follows a similar expression pattern to that of the PIN RNA/PIN RNA-overlapped exon pair (left and central panels); 9 pairs were identified in which the 3' exon of the protein-coding transcript (right panel) does not follow the pattern of tissue expression of the PIN RNA and the corresponding PIN RNA-overlapped exon (left and central panels); 6 pairs in which the PIN RNA (left panel) has an expression pattern inverted in relation to that of the PIN RNA-overlapped exon (central panel). Each line represents a genomic locus covered by three different types of probes (antisense PIN RNA, PIN RNA-overlapped protein-coding exon and 3' protein-coding exon). For each line, expression intensities among the three tissues were normalized within each type of probe and colored as a function of the number of standard deviations from the mean value