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 .

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.

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.

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.