A Tool to Design Bridging Oligos Used to Detect Pseudouridylation Sites on RNA after CMC Treatment.

Pseudouridylation is one of the most abundant modifications found in RNAs. To identify the Pseudouridylation sites (Psi) in RNAs, several techniques have been developed, but the most common and robust is the CMC (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide) treatment, which consists in the addition of an adduct on Psi that inhibits the reverse transcription. Here, we describe a turnkey method and a tool to design the bridging oligo (DBO), which is somewhat difficult to design. Finally, we propose a trouble-shooting guide to help users.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-11.

We are delighted to share with you our eleventh Journal Club and highlight some of the most interesting papers published recently [...].

Proteasome inhibition alters mitotic progression through the upregulation of centromeric α-Satellite RNAs.

Cell cycle progression requires control of the abundance of several proteins and RNAs over space and time to properly transit from one phase to the next and to ensure faithful genomic inheritance in daughter cells. The proteasome, the main protein degradation system of the cell, facilitates the establishment of a proteome specific to each phase of the cell cycle. Its activity also strongly influences transcription. Here, we detected the upregulation of repetitive RNAs upon proteasome inhibition in human cancer cells using RNA-seq. The effect of proteasome inhibition on centromeres was remarkable, especially on α-Satellite RNAs. We showed that α-Satellite RNAs fluctuate along the cell cycle and interact with members of the cohesin ring, suggesting that these transcripts may take part in the regulation of mitotic progression. Next, we forced exogenous overexpression and used gapmer oligonucleotide targeting to demonstrate that α-Sat RNAs have regulatory roles in mitosis. Finally, we explored the transcriptional regulation of α-Satellite DNA. Through in silico analyses, we detected the presence of CCAAT transcription factor-binding motifs within α-Satellite centromeric arrays. Using high-resolution three-dimensional immuno-FISH and ChIP-qPCR, we showed an association between the α-Satellite upregulation and the recruitment of the transcription factor NFY-A to the centromere upon MG132-induced proteasome inhibition. Together, our results show that the proteasome controls α-Satellite RNAs associated with the regulation of mitosis.

Systematic Identification and Functional Validation of New snoRNAs in Human Muscle Progenitors.

Small non-coding RNAs (sncRNAs) represent an important class of regulatory RNAs involved in the regulation of transcription, RNA splicing or translation. Among these sncRNAs, small nucleolar RNAs (snoRNAs) mostly originate from intron splicing in humans and are central to posttranscriptional regulation of gene expression. However, the characterization of the complete repertoire of sncRNAs in a given cellular context and the functional annotation of the human transcriptome are far from complete. Here, we report the large-scale identification of sncRNAs in the size range of 50 to 200 nucleotides without a priori on their biogenesis, structure and genomic origin in the context of normal human muscle cells. We provided a complete set of experimental validation of novel candidate snoRNAs by evaluating the prerequisites for their biogenesis and functionality, leading to their validation as genuine snoRNAs. Interestingly, we also found intergenic snoRNAs, which we showed are in fact integrated into candidate introns of unannotated transcripts or degraded by the Nonsense Mediated Decay pathway. Hence, intergenic snoRNAs represent a new type of landmark for the identification of new transcripts that have gone undetected because of low abundance or degradation after the release of the snoRNA.

Multiple information carried by RNAs: total eclipse or a light at the end of the tunnel?

The findings that an RNA is not necessarily either coding or non-coding, or that a precursor RNA can produce different types of mature RNAs, whether coding or non-coding, long or short, have challenged the dichotomous view of the RNA world almost 15 years ago. Since then, and despite an increasing number of studies, the diversity of information that can be conveyed by RNAs is rarely searched for, and when it is known, it remains largely overlooked in further functional studies. Here, we provide an update with prominent examples of multiple functions that are carried by the same RNA or are produced by the same precursor RNA, to emphasize their biological relevance in most living organisms. An important consequence is that the overall function of their locus of origin results from the balance between various RNA species with distinct functions and fates. The consideration of the molecular basis of this multiplicity of information is obviously crucial for downstream functional studies when the targeted functional molecule is often not the one that is believed.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-6.

We are delighted to share with you our sixth Journal Club and highlight some of the most interesting papers published recently [...].

Short intron-derived ncRNAs.

Introns represent almost half of the human genome, although they are eliminated from transcripts through RNA splicing. Yet, different classes of non-canonical miRNAs have been proposed to originate directly from intron splicing. Here, we considered the alternative splicing of introns as an interesting source of miRNAs, compatible with a developmental switch. We report computational prediction of new Short Intron-Derived ncRNAs (SID), defined as precursors of smaller ncRNAs like miRNAs and snoRNAs produced directly by splicing, and tested their dependence on each key factor in canonical or alternative miRNAs biogenesis (Drosha, DGCR8, DBR1, snRNP70, U2AF65, PRP8, Dicer, Ago2). We found that about half of predicted SID rely on debranching of the excised intron-lariat by the enzyme DBR1, as proposed for mirtrons. However, we identified new classes of SID for which miRNAs biogenesis may rely on intermingling between canonical and alternative pathways. We validated selected SID as putative miRNAs precursors and identified new endogenous miRNAs produced by non-canonical pathways, including one hosted in the first intron of SRA (Steroid Receptor RNA activator). Consistent with increased SRA intron retention during myogenic differentiation, release of SRA intron and its associated mature miRNA decreased in cells from healthy subjects but not from myotonic dystrophy patients with splicing defects.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-5.

We are delighted to share with you our fifth Journal Club and highlight some of the most interesting papers published recently.[...].

Mammalian introns: when the junk generates molecular diversity.

Introns represent almost half of the human genome, yet their vast majority is eliminated from eukaryotic transcripts through RNA splicing. Nevertheless, they feature key elements and functions that deserve further interest. At the level of DNA, introns are genomic segments that can shelter independent transcription units for coding and non-coding RNAs which transcription may interfere with that of the host gene, and regulatory elements that can influence gene expression and splicing itself. From the RNA perspective, some introns can be subjected to alternative splicing. Intron retention appear to provide some plasticity to the nature of the protein produced, its distribution in a given cell type and timing of its translation. Intron retention may also serve as a switch to produce coding or non-coding RNAs from the same transcription unit. Conversely, splicing of introns has been directly implicated in the production of small regulatory RNAs. Hence, splicing of introns also appears to provide plasticity to the type of RNA produced from a genetic locus (coding, non-coding, short or long). We addressed these aspects to add to our understanding of mechanisms that control the fate of introns and could be instrumental in regulating genomic output and hence cell fate.

"Pocket-sized RNA-Seq": A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest.

Currently, the discovery of new small ncRNAs requires high throughput methods even in the case of focused research on the regulation of specific genes or set of genes. We propose herein a simple, rapid, efficient, and cost effective method to clone and sequence single, yet unknown, small ncRNA. This technique that we called "Pocket-sized RNA-Seq" or psRNA-seq is based on in vitro transcription, RNA pull down and adapted RACE-PCR methods that allow its implementation using either available commercial kits or in-house reagents.

Identification of a dinucleotide signature that discriminates coding from non-coding long RNAs.

To date, the main criterion by which long ncRNAs (lncRNAs) are discriminated from mRNAs is based on the capacity of the transcripts to encode a protein. However, it becomes important to identify non-ORF-based sequence characteristics that can be used to parse between ncRNAs and mRNAs. In this study, we first established an extremely selective workflow to define a highly refined database of lncRNAs which was used for comparison with mRNAs. Then using this highly selective collection of lncRNAs, we found the CG dinucleotide frequencies were clearly distinct. In addition, we showed that the bias in CG dinucleotide frequency was conserved in human and mouse genomes. We propose that this sequence feature will serve as a useful classifier in transcript classification pipelines. We also suggest that our refined database of "bona fide" lncRNAs will be valuable for the discovery of other sequence characteristics distinct to lncRNAs.

Identification of potentially new bifunctional RNA based on genome-wide data-mining of alternative splicing events.

It is now evident that the transcriptional output of the genome is much more complex than estimates based on the number of protein-coding genes, and that non-coding RNA widely increase the source of regulatory molecules, a role previously ascribed to proteins. Furthermore, the recent characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, adds an additional degree of complexity. Based on the SRA (Steroid Receptor RNA Activator) model, where bifunctionality is regulated by alternative splicing, we hypothesized that similar cases, not yet formally tested experimentally, might exist. Using freely available data from high-throughput sequencing projects, we propose here a bioinformatical identification of mRNA whose ORF are disrupted by alternative splicing events, especially by intron retention, and potentially representing a cognate non-coding RNA. Our data-mining approach revealed that the human genome contains around 300 possibilities of potentially new bifunctional RNA.

A20-binding inhibitor of nuclear factor-kappaB (NF-kappaB)-2 (ABIN-2) is an activator of inhibitor of NF-kappaB (IkappaB) kinase alpha (IKKalpha)-mediated NF-kappaB transcriptional activity.

NF-κB transcription factors are pivotal players in controlling inflammatory and immune responses, as well as cell proliferation and apoptosis. Aberrant regulation of NF-κB and the signaling pathways that regulate its activity have been involved in various pathologies, particularly cancers, as well as inflammatory and autoimmune diseases. NF-κB activation is tightly regulated by the IκB kinase (IKK) complex, which is composed of two catalytic subunits IKKα and IKKß, and a regulatory subunit IKKγ/NEMO. Although IKKα and IKKß share structural similarities, IKKα has been shown to have distinct biological functions. However, the molecular mechanisms that modulate IKKα activity have not yet been fully elucidated. To understand better the regulation of IKKα activity, we purified IKKα-associated proteins and identified ABIN-2. Here, we demonstrate that IKKα and IKKß both interact with ABIN-2 and impair its constitutive degradation by the proteasome. Nonetheless, ABIN-2 enhances IKKα- but not IKKß-mediated NF-κB activation by specifically inducing IKKα autophosphorylation and kinase activity. Furthermore, we found that ABIN-2 serine 146 is critical for the ABIN-2-dependent IKKα transcriptional up-regulation of specific NF-κB target genes. These results imply that ABIN-2 acts as a positive regulator of NF-κB-dependent transcription by activating IKKα.

Steroid receptor RNA activator protein binds to and counteracts SRA RNA-mediated activation of MyoD and muscle differentiation.

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.

When one is better than two: RNA with dual functions.

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.

Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues.

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.

Increasing the relative expression of endogenous non-coding Steroid Receptor RNA Activator (SRA) in human breast cancer cells using modified oligonucleotides.

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.

Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase.

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.