Long intronic noncoding RNA transcription: expression noise or expression choice?

Recently, it was discovered that non-protein-coding RNAs (ncRNAs) represent the majority of the human transcripts. Regulatory role of many classes of ncRNAs is broadly recognized; however, long intronic ncRNAs have received little attention. In the past few years, evidence that intronic regions are key sources of regulatory ncRNAs has first appeared. Here we present an updated vision of the intronic ncRNA world, giving special attention to the long intronic ncRNAs. We summarize aspects of their expression pattern, evolutionary constraints, biogenesis, and responsiveness to physiological stimuli, and postulate their mechanisms of action. Deciphering nature's choice of different types of messages conveyed by ncRNAs will shed light on the RNA-based layer of regulatory processes in eukaryotic cells.

Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci.

It has been postulated that noncoding RNAs (ncRNAs) are involved in the posttranscriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. We show here that the complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney, and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, 22 RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human-mouse intronic ncRNAs was confirmed using strand-specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Androgen responsive intronic non-coding RNAs.

BACKGROUND: Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. RESULTS: Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5-5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation. CONCLUSION: Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program.

Identificacao e caracterizacao de transcritos humanos: novas famílias de pequenas GTPases e novos Longos RNAs intrônicos não-codificantes / Identification and caracterization of human transcripts: novel small GTPase gene families and novel Long Intronic non-coding RNAs

Terminado o sequenciamento do genoma humano, as atenções se voltaram para a determinação do conjunto completo de transcritos humanos. Diversos trabalhos sugerem que enquanto apenas uma pequena fração de mRNAs codificantes para proteína não é conhecida, existe um grande número de RNAs não-codificantes (ncRNAs) ainda não caracterizados. Nesse contexto, o presente trabalho visou explorar as informações de expressão gênica contidas em ESTs para identificar e caracterizar novos transcritos humanos. A busca genômica por membros de famílias gênicas relacionadas com câncer levou a identificação de novas pequenas GTPases, destacando uma subfamília que deve apresentar função supressora tumoral em próstata. Uma classe de ncRNAs longos, sem splicing, expressos antisenso a partir de regiões intrônicas foi descrita utilizando plataformas de microarrays, construídas pelo grupo, enriquecidas com seqüências sem anotação. O perfil de expressão de 23 ncRNAs intrônicos estava significativamente correlacionado com o grau de diferenciação de tumores de próstata (Gleason Score), e pode ser utilizado como candidato a marcador molecular de prognóstico. Um total de 39 ncRNAs intrônicos responderam à estimulação por andrógeno, apontando para um mecanismo regulatório da expressão intrônica por sinais fisiológicos hormonais. A biogênese da expressão intrônica parece ser complexa, pois uma fração não é transcrita pela RNA Polimerase II. A transcrição intrônica estava correlacionada com uso de exons em células tratadas com andrógeno. Assinaturas de expressão intrônica conservadas em tecidos humanos e de camundongos, e interações de transcritos intrônicos com proteínas regulatórias foram observadas. Este trabalho contribui com novas e originais evidências que dão apoio ao papel postulado para esses ncRNAs no controle fino do programa transcricional humano

As antisense RNA gets intronic.

Recent work describing the transcriptional output of the human genome points to the existence of a significant number of non-coding RNA transcripts coming from intronic regions, with a fraction of these being oriented antisense relative to the protein-coding mRNA of the known gene. In this article, we survey the main findings of the large-scale expression analysis projects that led to the identification of antisense intronic messages and which demonstrate their ubiquitous expression in the human genome. We review the current knowledge on long, unspliced, intronic antisense transcripts, a new class of non-coding RNAs, recently described by our group to be correlated with the degree of tumor differentiation in prostate cancer, which we postulate is involved in the fine tuning of gene expression in eukaryotes. Possible mechanisms of antisense intronic transcript biogenesis and function in gene expression regulation are discussed, as is their involvement in human diseases. While there is still no conclusive evidence demonstrating a functional role for these long, intronic antisense messages, the far-reaching implications of their existence for the mechanisms regulating gene expression certainly warrant further experimentation.

Antisense intronic non-coding RNA levels correlate to the degree of tumor differentiation in prostate cancer.

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P< or =0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT-PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.

RASL11A, member of a novel small monomeric GTPase gene family, is down-regulated in prostate tumors.

We performed a genome-wide search for novel loci encoding for Ras-related proteins based on the genome mapping coordinates of the cancer-derived EST dataset at GenBank. Partial sequences from two novel human genes were identified and subsequently used for full length transcript cloning. RASL11A and ARL9 belong to two novel subfamilies coding for small GTPases that we found to be highly conserved among eukaryotes. The Arl9/Arl10 subfamily displays a conserved interswitch toggle that places it evolutionarily closer to the Arf family. Rasl11 proteins are more closely related to the Ras branch of GTPases. All orthologues newly identified here exhibit an Asn residue in place of the highly conserved Thr35 of the G domain, suggesting that the universal switch mechanism of small GTPases may be structurally different in this subfamily. We determined by Northern blot that RASL11A is transcribed in several human tissues and that it is down-regulated in prostate tumors as measured by quantitative real-time PCR. These results highlight a previously uncharacterized subfamily of Ras-related genes that may have a tumor suppressor role in prostate cancer.