Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome.

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes.

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

Traveling with purpose: cell-to-cell transport of plant mRNAs.

Messenger RNAs (mRNAs) in multicellular organisms can act as signals transported cell-to-cell and over long distances. In plants, mRNAs traffic cell-to-cell via plasmodesmata (PDs) and over long distances via the phloem vascular system to control diverse biological processes - such as cell fate and tissue patterning - in destination organs. Research on long-distance transport of mRNAs in plants has made remarkable progress, including the cataloguing of many mobile mRNAs, characterization of mRNA features important for transport, identification of mRNA-binding proteins involved in their transport, and understanding of the physiological roles of mRNA transport. However, information on short-range mRNA cell-to-cell transport is still limited. This review discusses the regulatory mechanisms and physiological functions of mRNA transport at the cellular and whole plant levels.

mRNA nuclear export: how mRNA identity features distinguish functional RNAs from junk transcripts.

The division of the cellular space into nucleoplasm and cytoplasm promotes quality control mechanisms that prevent misprocessed mRNAs and junk RNAs from gaining access to the translational machinery. Here, we explore how properly processed mRNAs are distinguished from both misprocessed mRNAs and junk RNAs by the presence or absence of various 'identity features'.

Immunological characterization and diagnostic models of RNA N6-methyladenosine regulators in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia, and it displays both clinical and molecular variability. RNA N6-methyladenosine (m6A) regulators are involved in a wide range of essential cellular processes. In this study, we aimed to identify molecular signatures associated with m6A in Alzheimer's disease and use those signatures to develop a predictive model. We examined the expression patterns of m6A regulators and immune features in Alzheimer's disease using the GSE33000 dataset. We examined the immune cell infiltration and molecular groups based on m6A-related genes in 310 Alzheimer's disease samples. The WGCNA algorithm was utilized to determine differently expressed genes within each cluster. After evaluating the strengths and weaknesses of the random forest model, the support vector machine model, the generalized linear model, and eXtreme Gradient Boosting, the best machine model was selected. Methods such as nomograms, calibration curves, judgment curve analysis, and the use of independent data sets were used to verify the accuracy of the predictions made. Alzheimer's disease and non-disease Alzheimer's groups were compared to identify dysregulated m6A-related genes and activated immune responses. In Alzheimer's disease, two molecular clusters linked to m6A were identified. Immune infiltration analysis indicated substantial variation in protection between groups. Cluster 1 included processes like the Toll-like receptor signaling cascade, positive regulation of chromatin binding, and numerous malignancies; cluster 2 included processes like the cell cycle, mRNA transport, and ubiquitin-mediated proteolysis. With a lower residual and root mean square error and a larger area under the curve (AUC = 0.951), the Random forest machine model showed the greatest discriminative performance. The resulting random forest model was based on five genes, and it performed well (AUC = 0.894) on external validation datasets. Accuracy in predicting Alzheimer's disease subgroups was also shown by analyses of nomograms, calibration curves, and decision curves. In this research, we methodically outlined the tangled web of connections between m6A and AD and created a promising prediction model for gauging the correlation between m6A subtype risk and AD pathology.

Self-demixing of mRNA copies buffers mRNA:mRNA and mRNA:regulator stoichiometries.

Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.

The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function.

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.

Cross-linking mass spectrometric analysis of the endogenous TREX complex from Saccharomyces cerevisiae.

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In Saccharomyces cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.

Virus Infection and mRNA Nuclear Export.

Gene expression in eukaryotes begins with transcription in the nucleus, followed by the synthesis of messenger RNA (mRNA), which is then exported to the cytoplasm for its translation into proteins. Along with transcription and translation, mRNA export through the nuclear pore complex (NPC) is an essential regulatory step in eukaryotic gene expression. Multiple factors regulate mRNA export and hence gene expression. Interestingly, proteins from certain types of viruses interact with these factors in infected cells, and such an interaction interferes with the mRNA export of the host cell in favor of viral RNA export. Thus, these viruses hijack the host mRNA nuclear export mechanism, leading to a reduction in host gene expression and the downregulation of immune/antiviral responses. On the other hand, the viral mRNAs successfully evade the host surveillance system and are efficiently exported from the nucleus to the cytoplasm for translation, which enables the continuation of the virus life cycle. Here, we present this review to summarize the mechanisms by which viruses suppress host mRNA nuclear export during infection, as well as the key strategies that viruses use to facilitate their mRNA nuclear export. These studies have revealed new potential antivirals that may be used to inhibit viral mRNA transport and enhance host mRNA nuclear export, thereby promoting host gene expression and immune responses.

RNA trafficking and subcellular localization-a review of mechanisms, experimental and predictive methodologies.

RNA localization is essential for regulating spatial translation, where RNAs are trafficked to their target locations via various biological mechanisms. In this review, we discuss RNA localization in the context of molecular mechanisms, experimental techniques and machine learning-based prediction tools. Three main types of molecular mechanisms that control the localization of RNA to distinct cellular compartments are reviewed, including directed transport, protection from mRNA degradation, as well as diffusion and local entrapment. Advances in experimental methods, both image and sequence based, provide substantial data resources, which allow for the design of powerful machine learning models to predict RNA localizations. We review the publicly available predictive tools to serve as a guide for users and inspire developers to build more effective prediction models. Finally, we provide an overview of multimodal learning, which may provide a new avenue for the prediction of RNA localization.

An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport.

Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here, we show that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). We find that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. We additionally show that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. Our observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

Mechanisms of RNA export and nuclear retention.

With the identification of huge amount of noncoding RNAs in recent years, the concept of RNA localization has extended from traditional mRNA export to RNA export of mRNA and ncRNA as well as nuclear retention of ncRNA. This review aims to summarize the recent findings from studies on the mechanisms of export of different RNAs and nuclear retention of some lncRNAs in higher eukaryotes, with a focus on splicing-dependent TREX recruitment for the export of spliced mRNA and the sequence-dependent mechanism of mRNA export in the absence of splicing. In addition, evidence to support the involvement of m6 A modification in RNA export with the coordination between the methylase complex and TREX complex as well as sequence-dependent nuclear retention of lncRNA is recapitulated. Finally, a model of sequence-dependent RNA localization is proposed along with the many questions that remain to be answered. This article is categorized under: RNA Export and Localization > RNA Localization RNA Export and Localization > Nuclear Export/Import.

mRNA transport, translation, and decay in adult mammalian central nervous system axons.

Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knockout mice validate FMRP's role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the adult axonal transcriptome is stably maintained by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function in vivo.

POLDIP3: At the Crossroad of RNA and DNA Metabolism.

POLDIP3 was initially identified as a DNA polymerase delta (Pol δ) interacting protein almost twenty years ago. Intriguingly, it also interacts with proteins involved in a variety of RNA related biological processes, such as transcription, pre-mRNA splicing, mRNA export, and translation. Studies in recent years revealed that POLDIP3 also plays critical roles in disassembling genome wide R-loop formation and activating the DNA damage checkpoint in vivo. Here, we review the functions of POLDIP3 in various RNA and DNA related cellular processes. We then propose a unified model to illustrate how POLDIP3 plays such a versatile role at the crossroad of the RNA and DNA metabolism.

Kinetics of mRNA nuclear export regulate innate immune response gene expression.

The abundance and stimulus-responsiveness of mature mRNA is thought to be determined by nuclear synthesis, processing, and cytoplasmic decay. However, the rate and efficiency of moving mRNA to the cytoplasm almost certainly contributes, but has rarely been measured. Here, we investigated mRNA export rates for innate immune genes. We generated high spatio-temporal resolution RNA-seq data from endotoxin-stimulated macrophages and parameterized a mathematical model to infer kinetic parameters with confidence intervals. We find that the effective chromatin-to-cytoplasm export rate is gene-specific, varying 100-fold: for some genes, less than 5% of synthesized transcripts arrive in the cytoplasm as mature mRNAs, while others show high export efficiency. Interestingly, effective export rates do not determine temporal gene responsiveness, but complement the wide range of mRNA decay rates; this ensures similar abundances of short- and long-lived mRNAs, which form successive innate immune response expression waves.

An Evolved 5' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants.

Although the coat protein (CP) has a relevant role in the long-distance movement of alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), its precise function is not fully understood. Previous results showed that a specific interaction between the C termini of the movement protein (MP) and the cognate CP is required for systemic transport. Thus, we have performed a compensatory evolution experiment using an AMV RNA3 derivative defective in long-distance transport that carries a BMV MP lacking the C-terminal 48 residues and unable to interact with the AMV CP. After several passages, five independent evolution lineages were able to move long distance. The analysis of the viral RNA of these lineages showed the presence of three different modifications located exclusively at the 5' untranslated region (5' UTR). The three evolved 5' UTR variants accumulated comparable levels of viral RNA and CP but reduced the accumulation of virus particles and the affinity between the 5' UTR and the AMV CP. In addition, the evolved 5' UTR increased cell-to-cell transport for both the AMV RNA3 carrying the BMV MP and that carrying the AMV MP. Finally, the evolved 5' UTRs allowed the systemic transport of an AMV RNA3 carrying a CP mutant defective in virus particles and increased the systemic transport of several AMV RNA3 derivatives carrying different viral MPs associated with the 30K superfamily. Altogether, our findings indicate that virus particles are not required for the systemic transport of AMV but also that BMV MP is competent for the short- and long-distance transport without the interaction with the CP. IMPORTANCE The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). The incapability of the viral MP to interact with the CP triggered virus variants that evolved to reduce the formation of virus particles, probably to increase the accessibility of the MP to the viral progeny. Our results point to the idea that virus particles would not be necessary for the viral systemic transport but would be necessary for vector virus transmission. This idea is reinforced by the observation that heterologous MPs also increased the systemic transport of the AMV constructs that have reduced encapsidation capabilities.

RNA export through the nuclear pore complex is directional.

The changes occurring in mRNA organization during nucleo-cytoplasmic transport and export, are not well understood. Moreover, directionality of mRNA passage through the nuclear pore complex (NPC) has not been examined within individual NPCs. Here we find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after transcription and at the nuclear periphery. Compaction levels of nuclear transcripts can be modulated by varying levels of SR proteins and by changing genome organization. Nuclear mRNPs are mostly rod-shaped with distant 5'/3'-ends, although for some, the ends are in proximity. The latter is more abundant in the cytoplasm and can be modified by translation inhibition. mRNAs and lncRNAs exiting the NPC exhibit predominant 5'-first export. In some cases, several adjacent NPCs are engaged in export of the same mRNA suggesting 'gene gating'. Altogether, we show that the mRNP is a flexible structure during travels, with 5'-directionality during export.