ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.

Regulated Intron Removal Integrates Motivational State and Experience.

Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. We report that in the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. We posit that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.

The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

Nuclear retention coupled with sequential polyadenylation dictates post-transcriptional m6A modification in the nucleus.

N6-methyladenosine (m6A) modification is deemed to be co-transcriptionally installed on pre-mRNAs, thereby influencing various downstream RNA metabolism events. However, the causal relationship between m6A modification and RNA processing is often unclear, resulting in premature or even misleading generalizations on the function of m6A modification. Here, we develop 4sU-coupled m6A-level and isoform-characterization sequencing (4sU-m6A-LAIC-seq) and 4sU-GLORI to quantify the m6A levels for both newly synthesized and steady-state RNAs at transcript and single-base-resolution levels, respectively, which enable dissecting the relationship between m6A modification and alternative RNA polyadenylation. Unexpectedly, our results show that many m6A addition events occur post-transcriptionally, especially on transcripts with high m6A levels. Importantly, we find higher m6A levels on shorter 3' UTR isoforms, which likely result from sequential polyadenylation of longer 3' UTR isoforms with prolonged nuclear dwelling time. Therefore, m6A modification can also take place post-transcriptionally to intimately couple with other key RNA metabolism processes to establish and dynamically regulate epi-transcriptomics in mammalian cells.

PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells.

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors.

Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.

Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation.

Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.

Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns.

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.

Gene architecture directs splicing outcome in separate nuclear spatial regions.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

Motors, anchors, and connectors: orchestrators of organelle inheritance.

Organelle inheritance is a process whereby organelles are actively distributed between dividing cells at cytokinesis. Much valuable insight into the molecular mechanisms of organelle inheritance has come from the analysis of asymmetrically dividing cells, which transport a portion of their organelles to the bud while retaining another portion in the mother cell. Common principles apply to the inheritance of all organelles, although individual organelles use specific factors for their partitioning. Inheritance factors can be classified as motors, which are required for organelle transport; anchors, which immobilize organelles at distinct cell structures; or connectors, which mediate the attachment of organelles to motors and anchors. Here, we provide an overview of recent advances in the field of organelle inheritance and highlight how motor, anchor, and connector molecules choreograph the segregation of a multicopy organelle, the peroxisome. We also discuss the role of organelle population control in the generation of cellular diversity.

Immunosuppression by Mutated Calreticulin Released from Malignant Cells.

Mutations affecting exon 9 of the CALR gene lead to the generation of a C-terminally modified calreticulin (CALR) protein that lacks the KDEL endoplasmic reticulum (ER) retention signal and consequently mislocalizes outside of the ER where it activates the thrombopoietin receptor in a cell-autonomous fashion, thus driving myeloproliferative diseases. Here, we used the retention using selective hooks (RUSH) assay to monitor the trafficking of CALR. We found that exon-9-mutated CALR was released from cells in response to the biotin-mediated detachment from its ER-localized hook, in vitro and in vivo. Cellular CALR release was confirmed in suitable mouse models bearing exon-9-mutated hematopoietic systems or tumors. Extracellular CALR mediated immunomodulatory effects and inhibited the phagocytosis of dying cancer cells by dendritic cells (DC), thereby suppressing antineoplastic immune responses elicited by chemotherapeutic agents or by PD-1 blockade. Altogether, our results demonstrate paracrine immunosuppressive effects for exon-9-mutated CALR.

Histone retention preserves epigenetic marks during heat stress-induced transcriptional memory in plants.

Plants often experience recurrent stressful events, for example, during heat waves. They can be primed by heat stress (HS) to improve the survival of more severe heat stress conditions. At certain genes, sustained expression is induced for several days beyond the initial heat stress. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), but it is unclear how this is maintained for extended periods. Here, we determined histone turnover by measuring the chromatin association of HS-induced histone H3.3. Genome-wide histone turnover was not homogenous; in particular, H3.3 was retained longer at heat stress memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, methylation loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation, but not vice versa. Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally mediated epigenetic modifications.

BUD13 Promotes a Type I Interferon Response by Countering Intron Retention in Irf7.

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.

Nuclear TARBP2 Drives Oncogenic Dysregulation of RNA Splicing and Decay.

Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.

Regulation of anion-Na+ coordination chemistry in electrolyte solvates for low-temperature sodium-ion batteries.

High-performance sodium storage at low temperature is urgent with the increasingly stringent demand for energy storage systems. However, the aggravated capacity loss is induced by the sluggish interfacial kinetics, which originates from the interfacial Na+ desolvation. Herein, all-fluorinated anions with ultrahigh electron donicity, trifluoroacetate (TFA-), are introduced into the diglyme (G2)-based electrolyte for the anion-reinforced solvates in a wide temperature range. The unique solvation structure with TFA- anions and decreased G2 molecules occupying the inner sheath accelerates desolvation of Na+ to exhibit decreased desolvation energy from 4.16 to 3.49 kJ mol-1 and 24.74 to 16.55 kJ mol-1 beyond and below -20 °C, respectively, compared with that in 1.0 M NaPF6-G2. These enable the cell of Na||Na3V2(PO4)3 to deliver 60.2% of its room-temperature capacity and high capacity retention of 99.2% after 100 cycles at -40 °C. This work highlights regulation of solvation chemistry for highly stable sodium-ion batteries at low temperature.

Stimulus-specific remodeling of the neuronal transcriptome through nuclear intron-retaining transcripts.

The nuclear envelope has long been considered primarily a physical barrier separating nuclear and cytosolic contents. More recently, nuclear compartmentalization has been shown to have additional regulatory functions in controlling gene expression. A sizeable proportion of protein-coding mRNAs is more prevalent in the nucleus than in the cytosol, suggesting regulated mRNA trafficking to the cytosol, but the mechanisms underlying controlled nuclear mRNA retention remain unclear. Here, we provide a comprehensive map of the subcellular localization of mRNAs in mature mouse cortical neurons, and reveal that transcripts retained in the nucleus comprise the majority of stable intron-retaining mRNAs. Systematically probing the fate of nuclear transcripts upon neuronal stimulation, we found opposite effects on sub-populations of transcripts: while some are targeted for degradation, others complete splicing to generate fully mature mRNAs that are exported to the cytosol and mediate rapid increases in protein levels. Finally, different forms of stimulation mobilize distinct groups of intron-retaining transcripts, with this selectivity arising from the activation of specific signaling pathways. Overall, our findings uncover a cue-specific control of intron retention as a major regulator of acute remodeling of the neuronal transcriptome.

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain.

Intron branchpoint (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect the binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after the addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during cotranscriptional splicing in Plad-B using single-molecule intron tracking to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between the binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten the characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

GR-pKa: a message-passing neural network with retention mechanism for pKa prediction.

During the drug discovery and design process, the acid-base dissociation constant (pKa) of a molecule is critically emphasized due to its crucial role in influencing the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties and biological activity. However, the experimental determination of pKa values is often laborious and complex. Moreover, existing prediction methods exhibit limitations in both the quantity and quality of the training data, as well as in their capacity to handle the complex structural and physicochemical properties of compounds, consequently impeding accuracy and generalization. Therefore, developing a method that can quickly and accurately predict molecular pKa values will to some extent help the structural modification of molecules, and thus assist the development process of new drugs. In this study, we developed a cutting-edge pKa prediction model named GR-pKa (Graph Retention pKa), leveraging a message-passing neural network and employing a multi-fidelity learning strategy to accurately predict molecular pKa values. The GR-pKa model incorporates five quantum mechanical properties related to molecular thermodynamics and dynamics as key features to characterize molecules. Notably, we originally introduced the novel retention mechanism into the message-passing phase, which significantly improves the model's ability to capture and update molecular information. Our GR-pKa model outperforms several state-of-the-art models in predicting macro-pKa values, achieving impressive results with a low mean absolute error of 0.490 and root mean square error of 0.588, and a high R2 of 0.937 on the SAMPL7 dataset.

GRable Version 1.0: A Software Tool for Site-Specific Glycoform Analysis With Improved MS1-Based Glycopeptide Detection With Parallel Clustering and Confidence Evaluation With MS2 Information.

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).