RESUMEN
Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.
Asunto(s)
ADN/genética , Enciclopedias como Asunto , Genoma Humano/genética , Anotación de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética/genética , Transcriptoma/genética , Alelos , Línea Celular , ADN Intergénico/genética , Elementos de Facilitación Genéticos , Exones/genética , Perfilación de la Expresión Génica , Genes/genética , Genómica , Humanos , Poliadenilación/genética , Isoformas de Proteínas/genética , ARN/biosíntesis , ARN/genética , Edición de ARN/genética , Empalme del ARN/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ARNRESUMEN
Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.
Asunto(s)
ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/genética , Empalme Alternativo , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 22/genética , Clonación Molecular , Exones , Genoma Humano , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Isoformas de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.
Asunto(s)
Células/metabolismo , Redes Reguladoras de Genes/fisiología , ARN/fisiología , Transcriptoma/fisiología , Algoritmos , Proteínas Quimerinas/química , Proteínas Quimerinas/genética , Cromosomas Humanos Par 1/genética , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Humanos , Masculino , Análisis por Micromatrices/métodos , Modelos Biológicos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/genética , Isoformas de ARN/química , Isoformas de ARN/genética , Isoformas de ARN/metabolismo , Transcripción Genética/genética , Estudios de Validación como AsuntoRESUMEN
Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.