Long noncoding RNAs usher in a new era in the biology of enhancers.

Enhancer-associated long noncoding RNAs act over long distances and across chromosomes to activate transcription at distal promoters. Here, we address the latest advances made toward understanding the role of long noncoding RNA expression and the involvement of these RNAs in enhancer function through association with protein factors and modulation of chromatin structure.

N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules.

Stress granules are an integral part of the stress response that are formed from non-translating mRNAs aggregated with proteins. While much is known about stress granules, the factors that drive their mRNA localization are incompletely described. Modification of mRNA can alter the properties of the nucleobases and affect processes such as translation, splicing and localization of individual transcripts. Here, we show that the RNA modification N4-acetylcytidine (ac4C) on mRNA associates with transcripts enriched in stress granules and that stress granule localized transcripts with ac4C are specifically translationally regulated. We also show that ac4C on mRNA can mediate localization of the protein NOP58 to stress granules. Our results suggest that acetylation of mRNA regulates localization of both stress-sensitive transcripts and RNA-binding proteins to stress granules and adds to our understanding of the molecular mechanisms responsible for stress granule formation.

Long noncoding RNAs with enhancer-like function in human cells.

While the long noncoding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to decreased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in activation of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

Recent progress in miRNA biogenesis and decay.

MicroRNAs are a class of small regulatory RNAs that mediate regulation of protein synthesis by recognizing sequence elements in mRNAs. MicroRNAs are processed through a series of steps starting from transcription and primary processing in the nucleus to precursor processing and mature function in the cytoplasm. It is also in the cytoplasm where levels of mature microRNAs can be modulated through decay mechanisms. Here, we review the recent progress in the lifetime of a microRNA at all steps required for maintaining their homoeostasis. The increasing knowledge about microRNA regulation upholds great promise as therapeutic targets.

Altered m6A RNA methylation contributes to hippocampal memory deficits in Huntington's disease mice.

N6-methyladenosine (m6A) regulates many aspects of RNA metabolism and is involved in learning and memory processes. Yet, the impact of a dysregulation of post-transcriptional m6A editing on synaptic impairments in neurodegenerative disorders remains unknown. Here we investigated the m6A methylation pattern in the hippocampus of Huntington's disease (HD) mice and the potential role of the m6A RNA modification in HD cognitive symptomatology. m6A modifications were evaluated in HD mice subjected to a hippocampal cognitive training task through m6A immunoprecipitation sequencing (MeRIP-seq) and the relative levels of m6A-modifying proteins (FTO and METTL14) by subcellular fractionation and Western blot analysis. Stereotaxic CA1 hippocampal delivery of AAV-shFTO was performed to investigate the effect of RNA m6A dysregulation in HD memory deficits. Our results reveal a m6A hypermethylation in relevant HD and synaptic related genes in the hippocampal transcriptome of Hdh+/Q111 mice. Conversely, m6A is aberrantly regulated in an experience-dependent manner in the HD hippocampus leading to demethylation of important components of synapse organization. Notably, the levels of RNA demethylase (FTO) and methyltransferase (METTL14) were modulated after training in the hippocampus of WT mice but not in Hdh+/Q111 mice. Finally, inhibition of FTO expression in the hippocampal CA1 region restored memory disturbances in symptomatic Hdh+/Q111 mice. Altogether, our results suggest that a differential RNA methylation landscape contributes to HD cognitive symptoms and uncover a role of m6A as a novel hallmark of HD.

Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs.

miRNAs are small regulatory RNAs involved in the regulation of translation of target transcripts. miRNA biogenesis is a multistep process starting with the cleavage of the primary miRNA transcript in the nucleus by the Microprocessor complex. Endogenous processing of pri-miRNAs is challenging to study and the in vivo kinetics of this process is not known. Here, we present a method for determining the processing kinetics of pri-miRNAs within intact cells over time, using a pulse-chase approach to label transcribed RNA during 15 min, and follow the processing within a 1-hour window after labeling with bromouridine. We show that pri-miRNAs exhibit different processing kinetics ranging from fast over intermediate to slow processing, and we provide evidence that pri-miRNA processing can occur both cotranscriptionally and post-transcriptionally.

MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation.

MicroRNAs (miRNAs) are small RNAs that function as posttranscriptional regulators of gene expression. miRNAs affect a variety of signaling pathways, and impaired miRNA regulation may contribute to the development of cancer and other diseases. Here we show that miRNA miR-10a interacts with the 5' untranslated region of mRNAs encoding ribosomal proteins to enhance their translation. miR-10a alleviates translational repression of the ribosomal protein mRNAs during amino acid starvation and is required for their translational induction following anisomycin treatment or overexpression of RAS. We show that miR-10a binds immediately downstream of the regulatory 5'TOP motif and that the 5'TOP regulatory complex and miR-10a are functionally interconnected. The results show that miR-10a may positively control global protein synthesis via the stimulation of ribosomal protein mRNA translation and ribosome biogenesis and hereby affect the ability of cells to undergo transformation.

Noncoding RNAs and enhancers: complications of a long-distance relationship.

Spatial and temporal regulation of gene expression is achieved through instructions provided by the distal transcriptional regulatory elements known as enhancers. How enhancers transmit such information to their targets has been the subject of intense investigation. Recent advances in high throughput analysis of the mammalian transcriptome have revealed a surprising result indicating that a large number of enhancers are transcribed to noncoding RNAs. Although long noncoding RNAs were initially shown to confer epigenetic transcriptional repression, recent studies have uncovered a role for a class of such transcripts in gene-specific activation, often from distal genomic regions. In this review, we discuss recent findings on the role of long noncoding RNAs in transcriptional regulation, with an emphasis on new developments on the functional links between long noncoding RNAs and enhancers.

Predicting nuclear G-quadruplex RNA-binding proteins with roles in transcription and phase separation.

RNA-binding proteins are central for many biological processes and their characterization has demonstrated a broad range of functions as well as a wide spectrum of target structures. RNA G-quadruplexes are important regulatory elements occurring in both coding and non-coding transcripts, yet our knowledge of their structure-based interactions is at present limited. Here, using theoretical predictions and experimental approaches, we show that many chromatin-binding proteins bind to RNA G-quadruplexes, and we classify them based on their RNA G-quadruplex-binding potential. Combining experimental identification of nuclear RNA G-quadruplex-binding proteins with computational approaches, we build a prediction tool that assigns probability score for a nuclear protein to bind RNA G-quadruplexes. We show that predicted G-quadruplex RNA-binding proteins exhibit a high degree of protein disorder and hydrophilicity and suggest involvement in both transcription and phase-separation into membrane-less organelles. Finally, we present the G4-Folded/UNfolded Nuclear Interaction Explorer System (G4-FUNNIES) for estimating RNA G4-binding propensities at http://service.tartaglialab.com/new_submission/G4FUNNIES .

Targeting Polyadenylation for Retention of RNA at Chromatin.

The various steps of RNA polymerase II transcription, including transcription initiation, splicing, and termination, are interlinked and tightly coordinated. Efficient 3'end processing is defined by sequence motifs emerging in the nascent-transcribed RNA strand and the cotranscriptional binding of regulatory proteins. The processing of a mature 3'end consists of cleavage and polyadenylation and is coupled with RNA polymerase II transcription termination and the dissociation of the nascent RNA transcript from the chromatin-associated transcriptional template. The subcellular and subnuclear topological specificity of the various RNA species is important for their functions. For instance, the formation of RNA-binding protein interactions, critical for the final outcome of gene expression, may require the nucleoplasmic fully spliced and polyadenylated form of an RNA transcript. Thus, interfering with the critical step of transcription termination and 3'end formation provides a means for assaying the functional potential of a given RNA of interest.In this protocol, we describe a method for blocking 3'end processing of the nascent RNA transcript, by using RNase H-inactive antisense oligonucleotides targeting cleavage and polyadenylation, delivered via transient transfection in cell culture.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-7.

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

Metabolic Pulse-Chase RNA Labeling for pri-miRNA Processing Dynamics.

miRNA biogenesis is a multistep process starting with the cleavage of the primary miRNA transcript in the nucleus by the microprocessor complex. The pri-miRNA processing kinetics has a high impact on the final regulative role of the mature miRNAs on the expression of their target transcripts. Thus studying the in vivo kinetics of the miRNA biogenesis could give more insights into the contribution of each individual miRNA on regulation of gene expression. Here, we describe step by step a method to determine the processing kinetics of pri-miRNAs in vivo, using a pulse-chase approach that can be used in downstream applications such as qPCR or deep sequencing. We explain in detail the various aspects of this approach that can be applied to different mammalian cell types. The nature of this protocol allows the in vivo study of pri-miRNA processing kinetics in cells treated with different conditions, mutants, and/or cancer cell lines under physiological conditions.

Inhibiting Pri-miRNA Processing with Target Site Blockers.

The pri-miRNA processing is important for the final regulatory role of miRNAs on the expression of their target transcripts. The processing variability between pri-miRNAs can determine the final miRNA abundance better than primary transcription itself. Thus studying the in vivo pri-miRNA biogenesis could give more insights into the contribution of each individual miRNA on regulation of gene expression. Interfering processing of a specific pri-miRNA has been challenging due to the nature of the current RNA interfence methods. Here, we describe step by step a method to arrest processing of specific pri-miRNAs in vivo using LNA microRNA Target Site Blockers. We explain in detail the various aspects of this approach that can easily be applied to different mammalian cell types. The nature of this protocol allows the in vivo study of pri-miRNA processing and processing kinetics in cells treated with different conditions, mutants, and/or cancer cell lines under physiological conditions.

Transient N-6-Methyladenosine Transcriptome Sequencing Reveals a Regulatory Role of m6A in Splicing Efficiency.

Splicing efficiency varies among transcripts, and tight control of splicing kinetics is crucial for coordinated gene expression. N-6-methyladenosine (m6A) is the most abundant RNA modification and is involved in regulation of RNA biogenesis and function. The impact of m6A on regulation of RNA splicing kinetics is unknown. Here, we provide a time-resolved high-resolution assessment of m6A on nascent RNA transcripts and unveil its importance for the control of RNA splicing kinetics. We find that early co-transcriptional m6A deposition near splice junctions promotes fast splicing, while m6A modifications in introns are associated with long, slowly processed introns and alternative splicing events. In conclusion, we show that early m6A deposition specifies the fate of transcripts regarding splicing kinetics and alternative splicing.

Long ncRNA A-ROD activates its target gene DKK1 at its release from chromatin.

Long ncRNAs are often enriched in the nucleus and at chromatin, but whether their dissociation from chromatin is important for their role in transcription regulation is unclear. Here, we group long ncRNAs using epigenetic marks, expression and strength of chromosomal interactions; we find that long ncRNAs transcribed from loci engaged in strong long-range chromosomal interactions are less abundant at chromatin, suggesting the release from chromatin as a crucial functional aspect of long ncRNAs in transcription regulation of their target genes. To gain mechanistic insight into this, we functionally validate the long ncRNA A-ROD, which enhances DKK1 transcription via its nascent spliced released form. Our data provide evidence that the regulatory interaction requires dissociation of A-ROD from chromatin, with target specificity ensured within the pre-established chromosomal proximity. We propose that the post-transcriptional release of a subset of long ncRNAs from the chromatin-associated template plays an important role in their function as transcription regulators.

LincRNA H19 protects from dietary obesity by constraining expression of monoallelic genes in brown fat.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.

Cellular Fractionation and Isolation of Chromatin-Associated RNA.

In eukaryotic cells, the synthesis, processing, and functions of RNA molecules are confined to distinct subcellular compartments. Biochemical fractionation of cells prior to RNA isolation thus enables the analysis of distinct steps in the lifetime of individual RNA molecules that would be masked in bulk RNA preparations from whole cells. Here, we describe a simple two-step differential centrifugation protocol for the isolation of cytoplasmic, nucleoplasmic, and chromatin-associated RNA that can be used in downstream applications such as qPCR or deep sequencing. We discuss various aspects of this fractionation protocol, which can be readily applied to many mammalian cell types. For the study of long noncoding RNAs and enhancer RNAs in regulation of transcription especially the preparation of chromatin-associated RNA can contribute significantly to further developments.

LNA-modified oligonucleotides mediate specific inhibition of microRNA function.

microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus be applied to studies of miRNA functions and validation of putative target genes.

Bidirectional expression of long ncRNA/protein-coding gene pairs in cancer.

Bidirectional initiation of transcription by RNA polymerase II occurs prevalently at active promoters during protein-coding gene (PCG) expression. Upstream, antisense noncoding RNAs (ncRNAs) of differing lengths, stabilities and processings are being expressed from these promoters in concert with downstream, processive messenger RNA transcription. Although abundantly detected, the functional role and regulatory capacity of such transcripts have only been determined for individual cases. Long ncRNAs in general are reportedly able to regulate all steps of the gene expression process. Therefore, to get insight into the functionality of long ncRNAs transcribed bidirectionally from cancer-associated PCGs is of interest, as expression changes of tumor suppressor genes and oncogenes are prevalent in cancer.Here, we review the sources and characteristics of antisense transcription occurring at PCG loci in the human genome, and focus on the functional impact of bidirectional long ncRNA expression at cancer-associated PCGs.

Serial interactome capture of the human cell nucleus.

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.