Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of stem cell maintenance and neuronal differentiation.

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.

The lncRNA Malat1 is trafficked to the cytoplasm as a localized mRNA encoding a small peptide in neurons.

Synaptic function in neurons is modulated by local translation of mRNAs that are transported to distal portions of axons and dendrites. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is broadly expressed across cell types, almost exclusively as a nuclear long noncoding RNA. We found that in differentiating neurons, a portion of Malat1 RNA redistributes to the cytoplasm. Depletion of Malat1 using antisense oligonucleotides (ASOs) stimulates the expression of particular pre- and postsynaptic proteins, implicating Malat1 in their regulation. Neuronal Malat1 is localized in puncta of both axons and dendrites that costain with Staufen1 protein, similar to neuronal RNA granules formed by locally translated mRNAs. Ribosome profiling of cultured mouse cortical neurons identified ribosome footprints within a 5' region of Malat1 containing short open reading frames. The upstream-most reading frame (M1) of the Malat1 locus was linked to the GFP-coding sequence in mouse embryonic stem cells. When these gene-edited cells were differentiated into glutamatergic neurons, the M1-GFP fusion protein was expressed. Antibody staining for the M1 peptide confirmed its presence in wild-type neurons and showed that M1 expression was enhanced by synaptic stimulation with KCl. Our results indicate that Malat1 serves as a cytoplasmic coding RNA in the brain that is both modulated by and modulates synaptic function.

The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

The lncRNA Malat1 is trafficked to the cytoplasm as a localized mRNA encoding a small peptide in neurons.

Synaptic function is modulated by local translation of mRNAs that are transported to distal portions of axons and dendrites. The Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is broadly expressed across cell types, almost exclusively as a nuclear non-coding RNA. We found that in differentiating neurons, a portion of Malat1 RNA redistributes to the cytoplasm. Depletion of Malat1 from neurons stimulated expression of particular pre- and post- synaptic proteins, implicating Malat1 in their regulation. Neuronal Malat1 is localized to both axons and dendrites in puncta that co-stain with Staufen1 protein, similar to neuronal granules formed by locally translated mRNAs. Ribosome profiling of mouse cortical neurons identified ribosome footprints within a region of Malat1 containing short open reading frames. The upstream-most reading frame (M1) of the Malat1 locus was linked to the GFP coding sequence in mouse ES cells. When these gene-edited cells were differentiated into glutamatergic neurons, the M1-GFP fusion protein was expressed. Antibody staining for the M1 peptide confirmed its presence in wildtype neurons, and showed enhancement of M1 expression after synaptic stimulation with KCL. Our results indicate that Malat1 serves as a cytoplasmic coding RNA in the brain that is both modulated by and modulates synaptic function.

Aging differentially alters the transcriptome and landscape of chromatin accessibility in the male and female mouse hippocampus.

Aging-related memory impairment and pathological memory disorders such as Alzheimer's disease differ between males and females, and yet little is known about how aging-related changes in the transcriptome and chromatin environment differ between sexes in the hippocampus. To investigate this question, we compared the chromatin accessibility landscape and gene expression/alternative splicing pattern of young adult and aged mouse hippocampus in both males and females using ATAC-seq and RNA-seq. We detected significant aging-dependent changes in the expression of genes involved in immune response and synaptic function and aging-dependent changes in the alternative splicing of myelin sheath genes. We found significant sex-bias in the expression and alternative splicing of hundreds of genes, including aging-dependent female-biased expression of myelin sheath genes and aging-dependent male-biased expression of genes involved in synaptic function. Aging was associated with increased chromatin accessibility in both male and female hippocampus, especially in repetitive elements, and with an increase in LINE-1 transcription. We detected significant sex-bias in chromatin accessibility in both autosomes and the X chromosome, with male-biased accessibility enriched at promoters and CpG-rich regions. Sex differences in gene expression and chromatin accessibility were amplified with aging, findings that may shed light on sex differences in aging-related and pathological memory loss.

XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

The apoptotic splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing and spliceosome assembly is limited by technical challenges to examining spliceosomes in vivo. Here we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. Sequencing these pre-mRNA fragments allowed the transcriptome-wide mapping of branch sites with high sensitivity. In addition to known U2 snRNP proteins, these complexes contained the proteins RBM5 and RBM10. RBM5 and RBM10 are alternative splicing regulators that control exons affecting apoptosis and cell proliferation in cancer, but were not previously shown to associate with the U2 snRNP or to play roles in branch site selection. We delineate a common segment of RBM5 and RBM10, separate from their known functional domains, that is required for their interaction with the U2 snRNP. We identify a large set of splicing events regulated by RBM5 and RBM10 and find that they predominantly act as splicing silencers. Disruption of their U2 interaction renders the proteins inactive for repression of many alternative exons. We further find that these proteins assemble on branch sites of nearly all exons across the transcriptome, including those whose splicing is not altered by them. We propose a model where RBM5 and RBM10 act as components of the U2 snRNP complex. From within this complex, they sense structural features of branchpoint recognition to either allow progression to functional spliceosome or rejection of the complex to inhibit splicing.

The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells.

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells.

Fluorescence in situ hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. We present an improved method for FISH probe production that yields high-purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The degree of labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO, and Alexa dyes) was mostly >90%, comparable with commercial probes. The ease and low cost of production allowed the generation of probe sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1 Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Tracking pre-mRNA maturation across subcellular compartments identifies developmental gene regulation through intron retention and nuclear anchoring.

Steps of mRNA maturation are important gene regulatory events that occur in distinct cellular locations. However, transcriptomic analyses often lose information on the subcellular distribution of processed and unprocessed transcripts. We generated extensive RNA-seq data sets to track mRNA maturation across subcellular locations in mouse embryonic stem cells, neuronal progenitor cells, and postmitotic neurons. We find disparate patterns of RNA enrichment between the cytoplasmic, nucleoplasmic, and chromatin fractions, with some genes maintaining more polyadenylated RNA in chromatin than in the cytoplasm. We bioinformatically defined four regulatory groups for intron retention, including complete cotranscriptional splicing, complete intron retention in the cytoplasmic RNA, and two intron groups present in nuclear and chromatin transcripts but fully excised in cytoplasm. We found that introns switch their regulatory group between cell types, including neuronally excised introns repressed by polypyrimidine track binding protein 1 (PTBP1). Transcripts for the neuronal gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1) are highly expressed in mESCs but are absent from the cytoplasm. Instead, incompletely spliced Gabbr1 RNA remains sequestered on chromatin, where it is bound by PTBP1, similar to certain long noncoding RNAs. Upon neuronal differentiation, Gabbr1 RNA becomes fully processed and exported for translation. Thus, splicing repression and chromatin anchoring of RNA combine to allow posttranscriptional regulation of Gabbr1 over development. For this and other genes, polyadenylated RNA abundance does not indicate functional gene expression. Our data sets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development.

Publisher Correction: A protein assembly mediates Xist localization and gene silencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A protein assembly mediates Xist localization and gene silencing.

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear1. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus2 and binding diverse proteins3-5 to achieve X-chromosome inactivation (XCI)6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off8. Here we show that the Xist RNA-binding proteins PTBP19, MATR310, TDP-4311 and CELF112 assemble on the multivalent E-repeat element of Xist7 and, via self-aggregation and heterotypic protein-protein interactions, form a condensate1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase8, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

Pathway-guided analysis identifies Myc-dependent alternative pre-mRNA splicing in aggressive prostate cancers.

We sought to define the landscape of alternative pre-mRNA splicing in prostate cancers and the relationship of exon choice to known cancer driver alterations. To do so, we compiled a metadataset composed of 876 RNA-sequencing (RNA-Seq) samples from five publicly available sources representing a range of prostate phenotypes from normal tissue to drug-resistant metastases. We subjected these samples to exon-level analysis with rMATS-turbo, purpose-built software designed for large-scale analyses of splicing, and identified 13,149 high-confidence cassette exon events with variable incorporation across samples. We then developed a computational framework, pathway enrichment-guided activity study of alternative splicing (PEGASAS), to correlate transcriptional signatures of 50 different cancer driver pathways with these alternative splicing events. We discovered that Myc signaling was correlated with incorporation of a set of 1,039 cassette exons enriched in genes encoding RNA binding proteins. Using a human prostate epithelial transformation assay, we confirmed the Myc regulation of 147 of these exons, many of which introduced frameshifts or encoded premature stop codons. Our results connect changes in alternative pre-mRNA splicing to oncogenic alterations common in prostate and many other cancers. We also establish a role for Myc in regulating RNA splicing by controlling the incorporation of nonsense-mediated decay-determinant exons in genes encoding RNA binding proteins.

Deep-learning augmented RNA-seq analysis of transcript splicing.

A major limitation of RNA sequencing (RNA-seq) analysis of alternative splicing is its reliance on high sequencing coverage. We report DARTS (https://github.com/Xinglab/DARTS), a computational framework that integrates deep-learning-based predictions with empirical RNA-seq evidence to infer differential alternative splicing between biological samples. DARTS leverages public RNA-seq big data to provide a knowledge base of splicing regulation via deep learning, thereby helping researchers better characterize alternative splicing using RNA-seq datasets even with modest coverage.

Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse.

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem-loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

Author Correction: Inhibiting amyloid-ß cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design.

In the version of this Article originally published online, the upper right panel of Fig. 5a was mistakenly a repeat of the lower right panel. This has now been corrected in all versions of the Article.

Inhibiting amyloid-ß cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design.

Inhibiting the interaction between amyloid-ß (Aß) and a neuronal cell surface receptor, LilrB2, has been suggested as a potential route for treating Alzheimer's disease. Supporting this approach, Alzheimer's-like symptoms are reduced in mouse models following genetic depletion of the LilrB2 homologue. In its pathogenic, oligomeric state, Aß binds to LilrB2, triggering a pathway to synaptic loss. Here we identify the LilrB2 binding moieties of Aß (16KLVFFA21) and identify its binding site on LilrB2 from a crystal structure of LilrB2 immunoglobulin domains D1D2 complexed to small molecules that mimic phenylalanine residues. In this structure, we observed two pockets that can accommodate the phenylalanine side chains of KLVFFA. These pockets were confirmed to be 16KLVFFA21 binding sites by mutagenesis. Rosetta docking revealed a plausible geometry for the Aß-LilrB2 complex and assisted with the structure-guided selection of small molecule inhibitors. These molecules inhibit Aß-LilrB2 interactions in vitro and on the cell surface and reduce Aß cytotoxicity, which suggests these inhibitors are potential therapeutic leads against Alzheimer's disease.

Rbfox1 Regulates Synaptic Transmission through the Inhibitory Neuron-Specific vSNARE Vamp1.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.