The steroid receptor RNA activator (SRA) has the unusual property to function as both a non-coding RNA (ncRNA) and a protein SRAP. SRA ncRNA is known to increase the activity of a range of nuclear receptors as well as the master regulator of muscle differentiation MyoD. The contribution of SRA to either a ncRNA or a protein is influenced by alternative splicing of the first intron, the retention of which disrupts the SRAP open reading frame. We reported here that the ratio between non-coding and coding SRA isoforms increased during myogenic differentiation of human satellite cells but not myotonic dystrophy patient satellite cells, in which differentiation capacity is affected. Using constructs that exclusively produce SRA ncRNA or SRAP, we demonstrated that whereas SRA ncRNA was indeed an enhancer of myogenic differentiation and myogenic conversion of non-muscle cells through the co-activation of MyoD activity, SRAP prevented this SRA RNA-dependant co-activation. Interestingly, the SRAP inhibitory effect is mediated through the interaction of SRAP with its RNA counterpart via its RRM-like domain interacting with the functional sub-structure of SRA RNA, STR7. This study thus provides a new model for SRA-mediated regulation of MyoD transcriptional activity in the promotion of normal muscle differentiation, which takes into account the nature of SRA molecules present.

Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B.

Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues.DNA methylation | immunodeficiency | centromeric instability | facial anomalies | E2F family | hypomorphic mutation | hox genes

The Steroid Receptor RNA Activator 1 (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon- 1 extended SRA1 isoforms containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.

The steroid receptor RNA activator (SRA) has previously been characterized as belonging to the growing family of functional non-coding RNAs. However, we recently reported the Western blot detection of a putative endogenous SRA protein (SRAP) in breast cancer cells. Herein, we successfully suppressed the expression of this protein through specific RNA interference assay, unequivocally confirming its existence. Moreover, using database searches and Western blot analysis, we also showed that SRAP is highly conserved among chordata. Overall, our results suggest that SRA is the first example of a new class of functional RNAs also able to encode a protein.

The central dogma of biology, until not long ago, held that genetic information stored on DNA molecules was translated into the final protein products through RNA as intermediate molecules. Then, an additional level of complexity in the regulation of genome expression was added, implicating new classes of RNA molecules called non-coding RNA (ncRNA). These ncRNA are also often referred to as functional RNA in that, although they do not contain the capacity to encode proteins, do have a function as RNA molecules. They have been thus far considered as truly non-coding RNA since no ORF long enough to be considered, nor protein, have been associated with them. However, the recent identification and characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, suggests that a definite categorization of some RNA molecules is far from being straightforward. Indeed, several RNA primarily classified as non-protein-coding RNA has been showed to hold coding capacities and associated peptides. Conversely, mRNA, usually regarded as strictly protein-coding, may act as functional RNA molecules. Here, we describe several examples of these bifunctional RNA that have been already characterized from bacteria to mammals. We also extend this concept to fortuitous acquisition of dual function in pathological conditions and to the recently highlighted duality between information carried by a gene and its pseudogenes counterparts.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with nonsmall-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4 -120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P ¼ 0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P ¼ 0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P ¼ 0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC.

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Student's t-test, P < 0.003) higher SRA–intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA–intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.