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Summary: Transcription and export (TREX) is a multi‐subunit complex that links synthesis, processing and export of mRNAs. It interacts with the RNA helicase UAP56 and export factors such as MOS11 and ALYs to facilitate nucleocytosolic transport of mRNAs. Plant MOS11 is a conserved, but sparsely researched RNA‐binding export factor, related to yeast Tho1 and mammalian CIP29/SARNP.Using biochemical approaches, the domains of Arabidopsis thaliana MOS11 required for interaction with UAP56 and RNA‐binding were identified. Further analyses revealed marked genetic interactions between MOS11 and ALY genes. Cell fractionation in combination with transcript profiling demonstrated that MOS11 is required for export of a subset of mRNAs that are shorter and more GC‐rich than MOS11‐independent transcripts.The central α‐helical domain of MOS11 proved essential for physical interaction with UAP56 and for RNA‐binding. MOS11 is involved in the nucleocytosolic transport of mRNAs that are upregulated under stress conditions and accordingly mos11 mutant plants turned out to be sensitive to elevated NaCl concentrations and heat stress.Collectively, our analyses identify functional interaction domains of MOS11. In addition, the results establish that mRNA export is critically involved in the plant response to stress conditions and that MOS11 plays a prominent role at this. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) interact with mRNA to form supramolecular complexes called messenger ribonucleoprotein (mRNP) particles. These dynamic assemblies direct and regulate individual steps of gene expression; however, their composition and functional importance remain largely unknown. Here, we develop a total internal reflection fluorescence-based single-molecule imaging assay to investigate stoichiometry and co-occupancy of 15 RBPs within mRNPs from Saccharomyces cerevisiae. We show compositional heterogeneity of single mRNPs and plasticity across different growth conditions, with major co-occupants of mRNPs containing the nuclear cap-binding complex identified as Yra1 (1-10 copies), Nab2 (1-6 copies), and Npl3 (1-6 copies). Multicopy Yra1-bound mRNPs are specifically co-occupied by the THO complex and assembled on mRNAs biased by transcript length and RNA secondary structure. Yra1 depletion results in decreased compaction of nuclear mRNPs demonstrating a packaging function. Together, we provide a quantitative framework for gene- and condition-dependent RBP occupancy and stoichiometry in individual nuclear mRNPs.

Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no‐ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A‐modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold between interferon‐stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination‐mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A‐tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness‐related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A‐tagged mRNA associated with stemness maintenance, which suppresses BCSCs self‐renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A‐modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A‐tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy. [ABSTRACT FROM AUTHOR]

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae (baker´s yeast) has yielded relevant in-sights into some of the basic mechanisms of organismal aging. Among these are genomic instability, oxidative stress, caloric restriction and mitochondrial dysfunction. Several genes are known to have an impact on the aging process, with corresponding mutants exhibiting short- or long-lived phenotypes. Re-search dedicated to unraveling the underlying cellular mechanisms can sup-port the identification of conserved mechanisms of aging in other species. One of the hitherto less studied fields in yeast aging is how the organism regulates its gene expression at the transcriptional level. To our knowledge, we present the first investigation into alternative splicing, particularly intron retention, during replicative aging of S. cerevisiae. This was achieved by utiliz-ing the IRFinder algorithm on a previously published RNA-seq data set by Janssens et al. (2015). In the present work, 44 differentially retained introns in 43 genes were identified during replicative aging. We found that genes with altered intron retention do not display significant changes in overall transcript levels. It was possible to functionally assign distinct groups of these genes to the cellular processes of mRNA processing and export (e.g., YRA1) in early and middle-aged yeast, and protein ubiquitination (e.g., UBC5) in older cells. In summary, our work uncovers a previously unexplored layer of the transcrip-tional program of yeast aging and, more generally, expands the knowledge on the occurrence of alternative splicing in baker´s yeast.

The eukaryotic translation initiation factor eIF4E acts as a multifunctional factor that simultaneously influences mRNA processing, export, and translation in many organisms. Its multifactorial effects are derived from its capacity to bind to the methyl-7-guanosine cap on the 5’end of mRNAs and thus can act as a cap chaperone for transcripts in the nucleus and cytoplasm. In this review, we describe the multifactorial roles of eIF4E in major mRNA-processing events including capping, splicing, cleavage and polyadenylation, nuclear export and translation. We discuss the evidence that eIF4E acts at two levels to generate widescale changes to processing, export and ultimately the protein produced. First, eIF4E alters the production of components of the mRNA processing machinery, supporting a widescale reprogramming of multiple mRNA processing events. In this way, eIF4E can modulate mRNA processing without physically interacting with target transcripts. Second, eIF4E also physically interacts with both capped mRNAs and components of the RNA processing or translation machineries. Further, specific mRNAs are sensitive to eIF4E only in particular mRNA processing events. This selectivity is governed by the presence of cis-acting elements within mRNAs known as USER codes that recruit relevant co-factors engaging the appropriate machinery. In all, we describe the molecular bases for eIF4E’s multifactorial function and relevant regulatory pathways, discuss the basis for selectivity, present a compendium of ~80 eIF4E-interacting factors which play roles in these activities and provide an overview of the relevance of its functions to its oncogenic potential. Finally, we summarize early-stage clinical studies targeting eIF4E in cancer.

The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm. [ABSTRACT FROM AUTHOR]

Recent findings position the eukaryotic translation initiation factor eIF4E as a novel modulator of mRNA splicing, a process that impacts the form and function of resultant proteins. eIF4E physically interacts with the spliceosome and with some intron‐containing transcripts implying a direct role in some splicing events. Moreover, eIF4E drives the production of key components of the splicing machinery underpinning larger scale impacts on splicing. These drive eIF4E‐dependent reprogramming of the splicing signature. This work completes a series of studies demonstrating eIF4E acts in all the major mRNA maturation steps whereby eIF4E drives production of the RNA processing machinery and escorts some transcripts through various maturation steps. In this way, eIF4E couples the mRNA processing‐export‐translation axis linking nuclear mRNA processing to cytoplasmic translation. eIF4E elevation is linked to worse outcomes in acute myeloid leukemia patients where these activities are dysregulated. Understanding these effects provides new insight into post‐transcriptional control and eIF4E‐driven cancers. [ABSTRACT FROM AUTHOR]

Abstract Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no‐ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A‐modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold between interferon‐stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination‐mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A‐tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness‐related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A‐tagged mRNA associated with stemness maintenance, which suppresses BCSCs self‐renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A‐modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A‐tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy.

Following the transcription step, the newly synthesized mRNA is exported from the nucleus to the cytoplasm and further to the translation site. The TREX-2 complex is involved in the step of mRNA export from the nucleus to the cytoplasm. This complex in Drosophila melanogaster consists of four proteins: Xmas-2, PCID2, ENY2, and Sem1p. In our work, we have shown that deletion of the C-terminal sequence of PCID2 leads to a decrease in the interaction of the protein with RNA and to impaired mRNA export from the nucleus to the cytoplasm in D. melanogaster. [ABSTRACT FROM AUTHOR]

The TREX-2 protein complex is the key complex involved in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a joint protein complex of TREX-2 with ORC was isolated in D. melanogaster. It was shown that the interaction of TREX-2 with ORC is necessary for efficient mRNA export from the nucleus to the cytoplasm. In this work, we showed that the TREX-2-ORC joint complex is also formed in human cells. [ABSTRACT FROM AUTHOR]

Nucleopore Complexes are intricate protein assemblies composed of diverse nucleoporins, which serve as crucial mediators for the bidirectional movement of molecules between the nucleus and cytosol. These nucleoporins share both structural and functional characteristics across yeast, mammals, and plants. This review highlights these shared architectural elements and further examines specific nucleoporins. A particular emphasis is placed on the putative homologs yeast NUP1, human NUP153, and plant NUP136, and their shared involvement in critical processes such as developmental coordination, gene regulation, and immune responses. Despite variations in their amino acid sequences, these proteins exhibit a notable degree of structural conservation, supporting a convergent evolution that would underlie their similar functionalities. [ABSTRACT FROM AUTHOR]

Cells must maintain a pool of processed and charged transfer RNAs (tRNA) to sustain translation capacity and efficiency. Numerous parallel pathways support the processing and directional movement of tRNA in and out of the nucleus to meet this cellular demand. Recently, several proteins known to control messenger RNA (mRNA) transport were implicated in tRNA export. The DEAD-box Protein 5, Dbp5, is one such example. In this study, genetic and molecular evidence demonstrates that Dbp5 functions parallel to the canonical tRNA export factor Los1. In vivo co-immunoprecipitation data further shows Dbp5 is recruited to tRNA independent of Los1, Msn5 (another tRNA export factor), or Mex67 (mRNA export adaptor), which contrasts with Dbp5 recruitment to mRNA that is abolished upon loss of Mex67 function. However, as with mRNA export, overexpression of Dbp5 dominant-negative mutants indicates a functional ATPase cycle and that binding of Dbp5 to Gle1 is required by Dbp5 to direct tRNA export. Biochemical characterization of the Dbp5 catalytic cycle demonstrates the direct interaction of Dbp5 with tRNA (or double-stranded RNA) does not activate Dbp5 ATPase activity, rather tRNA acts synergistically with Gle1 to fully activate Dbp5. These data suggest a model where Dbp5 directly binds tRNA to mediate export, which is spatially regulated via Dbp5 ATPase activation at nuclear pore complexes by Gle1.

The expression of bromodomain-containing proteins that regulate chromatin structure and accessibility must be tightly controlled to ensure the appropriate regulation of gene expression. In the yeast S. cerevisiae, Bromodomain Factor 2 (BDF2) expression is extensively regulated post-transcriptionally during stress by RNase III-mediated decay (RMD), which is triggered by cleavage of the BDF2 mRNA in the nucleus by the RNase III homolog Rnt1p. Previous studies have shown that RMD-mediated down-regulation of BDF2 is hyperactivated in osmotic stress conditions, yet the mechanisms driving the enhanced nuclear cleavage of BDF2 RNA under these conditions remain unknown. Here, we show that RMD hyperactivation can be detected in multiple stress conditions that inhibit mRNA export, and that Rnt1p remains primarily localized in the nucleus during salt stress. We show that globally inhibiting mRNA nuclear export by anchoring away mRNA biogenesis or export factors out of the nucleus can recapitulate RMD hyperactivation in the absence of stress. RMD hyperactivation requires Rnt1p nuclear localization but does not depend on the BDF2 gene endogenous promoter, and its efficiency is affected by the structure of the stem-loop cleaved by Rnt1p. Because multiple stress conditions have been shown to mediate global inhibition of mRNA export, our results suggest that the hyperactivation of RMD is primarily the result of the increased nuclear retention of the BDF2 mRNA during stress.

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Cajal bodies (CBs) are major sub-nuclear structures in most eucaryotic cells. In human adenovirus 5 (Ad5) infection, CBs are reorganized into microfoci in the late phase of infection. Here we show that many CB protein components (p80-coilin, SMN-1, and WRAP53) remained stable throughout most of the infectious cycle of Ad type 5 (Ad5) in human A549 epithelial cells, even when CBs were reorganized into microfoci. Reduction of p80-coilin expression by RNA interference resulted in significant reductions in the levels of early (E1A, E2A-DBP), intermediate (pIX and IVa2), and late (L1-IIIa, L2-penton base, L3-hexon, L4-100K, and L5-fiber) proteins in Ad5-infected A549 cells. Depletion of p80-coilin did not significantly alter the total cellular levels of the corresponding Ad cytoplasmic mRNAs (with the exception of E1A 12S and 13S and pIX mRNA) or the production of the Ad5 pre-mRNAs tested (E1A, E2A-DBP, IVa2, or late pre-mRNAs containing the tripartite leader). However, the ratio of viral cytoplasmic to nuclear-spliced Ad RNAs was reduced in p80-coilin-depleted, Ad5-infected cells compared to control-infected cells. Immunofluorescent staining of Ad5-infected cells revealed co-localization of p80-coilin with areas of immunoreactivity defined by a polyclonal antibody that recognized the L4-22K and L4-33K proteins in a fraction of microfoci. Immunoprecipitation analysis showed that only the L4-22K protein formed a stable complex with p80-coilin in Ad5-infected cells and in cells co-transfected with plasmids encoding p80-coilin and either the L4-22K or L4-33K protein. p80-coilin therefore plays an important role in Ad replication by complex formation with L4-22K and facilitating nuclear export of Ad mRNAs. IMPORTANCE The architecture of sub-nuclear structures of eucaryotic cells is often changed during the infectious cycle of many animal and plant viruses. Cajal bodies (CBs) form a major sub-nuclear structure whose functions may include the regulation of cellular RNA metabolism. During the lifecycle of human adenovirus 5 (Ad5), CBs are reorganized from their spherical-like structure into smaller clusters termed microfoci. The mechanism of this reorganization and its significance for virus replication has yet to be established. Here we show that the major CB protein, p80-coilin, facilitates the nuclear export of Ad5 transcripts. Depletion of p80-coilin by RNA interference led to lowered levels of viral proteins and infectious virus. p80-coilin was found to form a complex with the viral L4-22K protein in Ad5-infected cells and in some reorganized microfoci. These findings assign a new role for p80-coilin as a potential regulator of infection by a human DNA virus.

Cell viability largely depends on the surveillance of mRNA export and translation. Upon pre-mRNA processing and nuclear quality control, mature mRNAs are exported into the cytoplasm via Mex67-Mtr2 attachment. At the cytoplasmic site of the nuclear pore complex, the export receptor is displaced by the action of the DEAD-box RNA helicase Dbp5. Subsequent quality control of the open reading frame requires translation. Our studies suggest an involvement of Dbp5 in cytoplasmic no-go-and non-stop decay. Most importantly, we have also identified a key function for Dbp5 in translation termination, which identifies this helicase as a master regulator of mRNA expression. [ABSTRACT FROM AUTHOR]

The DEAD-box protein (DBP) Dbp5, a member of the superfamily II (SFII) helicases, has multiple reported roles in gene expression. First identified as an essential regulator of mRNA export in Saccharomyces cerevisiae, the enzyme now has reported functions in non-coding RNA export, translation, transcription, and DNA metabolism. Localization of the protein to various cellular compartments (nucleoplasm, nuclear envelope, and cytoplasm) highlights the ability of Dbp5 to modulate different stages of the RNA lifecycle. While Dbp5 has been well studied for > 20 years, several critical questions remain regarding the mechanistic principles that govern Dbp5 localization, substrate selection, and functions in gene expression. This review aims to take a holistic view of the proposed functions of Dbp5 and evaluate models that accommodate current published data.

ZC3H11A (zinc finger CCCH domain-containing protein 11A) is a stress-induced mRNA-binding protein required for efficient growth of nuclear-replicating viruses. The cellular functions of ZC3H11A during embryonic development are unknown. Here, we report the generation and phenotypic characterization of Zc3h11a knockout (KO) mice. Heterozygous null Zc3h11a mice were born at the expected frequency without distinguishable phenotypic differences compared with wild-type mice. In contrast, homozygous null Zc3h11a mice were missing, indicating that Zc3h11a is crucial for embryonic viability and survival. Zc3h11a -/- embryos were detected at the expected Mendelian ratios up to late preimplantation stage (E4.5). However, phenotypic characterization at E6.5 revealed degeneration of Zc3h11a -/- embryos, indicating developmental defects around the time of implantation. Transcriptomic analyses documented a dysregulation of glycolysis and fatty acid metabolic pathways in Zc3h11a-/- embryos at E4.5. Proteomic analysis indicated a tight interaction between ZC3H11A and mRNA-export proteins in embryonic stem cells. CLIP-seq analysis demonstrated that ZC3H11A binds a subset of mRNA transcripts that are critical for metabolic regulation of embryonic cells. Furthermore, embryonic stem cells with an induced deletion of Zc3h11a display an impaired differentiation toward epiblast-like cells and impaired mitochondrial membrane potential. Altogether, the results show that ZC3H11A is participating in export and posttranscriptional regulation of selected mRNA transcripts required to maintain metabolic processes in embryonic cells. While ZC3H11A is essential for the viability of the early mouse embryo, inactivation of Zc3h11a expression in adult tissues using a conditional KO did not lead to obvious phenotypic defects. [ABSTRACT FROM AUTHOR]

Directed migration of neural stem cells (NSCs) is critical for embryonic neurogenesis and the healing of neurological injuries. The long noncoding RNA (lncRNA) Pnky has been reported to regulate neuronal differentiation of NSCs by interacting with PTBP1. However, its regulatory effect on NSC migration remains to be determined. Herein, we identified that Pnky is also a key regulator of NSC migration in mice, as underscored by the finding that Pnky silencing suppressed but Pnky overexpression promoted the in vitro migration of both C17.2 and NE4C murine NSCs. Additionally, in vivo cell tracking demonstrated that Pnky depletion attenuated but Pnky overexpression facilitated the migration of NE4C cells in the spinal canal after transplantation via injection into the spinal canal. Mechanistically, Pnky regulated the expression of a core set of critical regulators that direct NSC migration, including MMP2, MMP9, Connexin43, Paxillin, AKT, ERK, and P38MAPK. Using catRAPID, a web server for large-scale prediction of protein–RNA interactions, the splicing factors U2AF1 and U2AF1L4, as well as the mRNA export adaptors SARNP, Aly/Ref, and THOC7, were predicted to interact strongly with Pnky. Further investigations using colocalization and RNA immunoprecipitation (RIP) assays confirmed the direct binding of Pnky to U2AF1, SARNP, Aly/Ref, and THOC7. Transcriptomic profiling revealed that as many as 5319 differential splicing events of 3848 genes, which were highly enriched in focal adhesion, PI3K-Akt and MAPK signaling pathways, were affected by Pnky depletion. The predominant subtype of differential splicing by Pnky depletion is intron retention, followed by alternative 5' and 3' splice sites and mutually exclusive exons. Moreover, Pnky knockdown substantially blocked but Pnky overexpression facilitated the export of MMP2, Paxillin, AKT, p38MAPK, and other mRNAs to the cytosol. Collectively, our data showed that through interacting with U2AF1, SARNP, Aly/Ref, and THOC7, Pnky couples and modulates the splicing and export of target mRNAs, which consequently controlling NSC migration. These findings provide a possible theoretical basis of NSC migration regulation. [ABSTRACT FROM AUTHOR]

The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster, TREX-2 consists of four major proteins: Xmas-2, ENY2, PCID2, and Sem1p. The Xmas-2 protein is the core subunit of the complex, with which other TREX-2 subunits interact. Xmas-2 homologues were found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein, can undergo cleavage into two parts, probably during apoptosis. We showed that the Xmas-2 protein of D. melanogaster can also split into two fragments. The resulting fragments of the protein correspond to the two large Xmas-2 domains. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster. [ABSTRACT FROM AUTHOR]

Despite extensive research on how mRNAs are quality controlled prior to export into the cytoplasm, the exact underlying mechanisms are still under debate. Specifically, it is unclear how quality control proteins at the entry of the nuclear pore complex (NPC) distinguish normal and aberrant mRNAs. While some of the involved components are suggested to act as switches and recruit different factors to normal versus aberrant mRNAs, some experimental and computational evidence suggests that the combined effect of the regulated stochastic interactions between the involved components could potentially achieve an efficient quality control of mRNAs. In this review, we present a state-of-the-art portrait of the mRNA quality control research and discuss the current hypotheses proposed for dynamics of the cooperation between the involved components and how it leads to their shared goal: mRNA quality control prior to export into the cytoplasm.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Export of RNA from the nucleus is essential for all eukaryotic cells and has emerged as a major step in the control of gene expression. mRNA molecules are required to complete a complex series of processing events and pass a quality control system to protect the cytoplasm from the translation of aberrant proteins. Many of these events are highly conserved across eukaryotes, reflecting their ancient origin, but significant deviation from a canonical pathway as described from animals and fungi has emerged in the trypanosomatids. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may also reveal additional, previously unsuspected, mRNA export pathways. mRNA export is a highly complex process at the core of gene expression controlling mRNA abundance. Steps include maturation, quality control ensuring that mRNAs are correctly coding, and the export process itself. Trypanosomes have distinct mechanisms for producing mRNA which sets them apart from animals and fungi. These include trans -splicing, absence of introns within open reading frames and polycistronic transcription. These require distinct processes to produce mature mRNAs, which are near identical in structure to higher eukaryotes. We describe how distinct mechanisms for mRNA processing have resulted in changes to mRNA export mechanisms, encompassing nuclear pore complex composition and organization, mRNA maturation, and quality control mechanisms. [ABSTRACT FROM AUTHOR]

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.

The maturation and export of mRNA from the nucleus through the nuclear pore complex is critical for maintaining an appropriate proteome in all eukaryotic cells. Here we summarize a previously unpublished screen in S. cerevisiae that utilized an established dT50 in situ hybridization assay to identify cold-sensitive mutants that accumulated bulk poly A RNA in the nucleus. The screen identified seven mutants in six complementation groups, including the brr6-1 strain that we described previously. In addition to brr6-1, we identified novel alleles of the key transport gene GLE1 and NUP188, a component of the Nic96 nucleoporin complex. Notably, we show that the nup188-brr7 allele causes defects in select protein import pathways as well as mRNA export. Given recent structural and functional evidence linking the Nic96 complex to transport components, this mutant may be particularly useful to the transport community.

Many negative-sense RNA viruses, including the highly pathogenic Ebola virus (EBOV), use cytoplasmic inclusion bodies (IBs) for viral RNA synthesis. However, it remains unclear how viral mRNAs are exported from these IBs for subsequent translation. We recently demonstrated that the nuclear RNA export factor 1 (NXF1) is involved in a late step in viral protein expression, i.e., downstream of viral mRNA transcription, and proposed it to be involved in this mRNA export process. We now provide further evidence for this function by showing that NXF1 is not required for translation of viral mRNAs, thus pinpointing its function to a step between mRNA transcription and translation. We further show that RNA binding of both NXF1 and EBOV NP is necessary for export of NXF1 from IBs, supporting a model in which NP hands viral mRNA over to NXF1 for export. Mapping of NP-NXF1 interactions allowed refinement of this model, revealing two separate interaction sites, one of them directly involving the RNA binding cleft of NP, even though these interactions are RNA-independent. Immuno-fluorescence analyses demonstrated that individual NXF1 domains are sufficient for its recruitment into IBs, and complementation assays helped to define NXF1 domains important for its function in the EBOV life cycle. Finally, we show that NXF1 is also required for protein expression of other viruses that replicate in cytoplasmic IBs, including Lloviu and Junín virus. These data suggest a role for NXF1 in viral mRNA export from IBs for various viruses, making it a potential target for broadly active antivirals. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm, and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyltransferases (KATs) promote transcription through acetylation of chromatin‐associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX‐2) complex, to the nuclear basket. The Esa1‐mediated nuclear export of mRNAs in turn promotes entry into S phase, which is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism is not only limited to G1/S‐expressed genes but also inhibits the expression of the nutrient‐regulated GAL1 gene specifically in daughter cells. Overall, these results reveal how acetylation can contribute to the functional plasticity of NPCs in mother and daughter yeast cells. In addition, our work demonstrates dual gene expression regulation by the evolutionarily conserved NuA4 complex, at the level of transcription and at the stage of mRNA export by modifying the nucleoplasmic entrance to nuclear pores. Synopsis: Lysine acetyltransferases (KATs) are known to associate with the nuclear pore complex (NPC), yet their role in regulating gene expression at this location is not fully understood. Here, in addition to regulating transcription, the budding yeast, KAT Esa1 (NuA4 complex), is found to also promote mRNA export. Esa1, the catalytic subunit of the NuA4 complex, acetylates the nuclear pore complex component Nup60Acetylation of Nup60 recruits the TREX‐2 mRNA export complex to the nuclear basketEsa1‐mediated mRNA export in G1 cells promotes their entry into S phaseDeacetylation of Nup60 by the daughter‐cell localized lysine deacetylase Hos3 causes causes displacement of mRNA export complexes and delays the G1/S transition in daughter cells [ABSTRACT FROM AUTHOR]

In eukaryotic cells, gene expression begins with transcription in the nucleus, followed by the maturation of messenger RNAs (mRNAs). These mRNA molecules are then exported to the cytoplasm through the nuclear pore complex (NPC), a process that serves as a critical regulatory phase of gene expression. The export of mRNA is intricately linked to precursor mRNA (pre-mRNA) processing, ensuring that only properly processed mRNA reaches the cytoplasm. This coordination is essential, as recent studies have revealed that mRNA export factors not only assist in transport but also influence upstream processing steps, adding a layer of complexity to gene regulation. Furthermore, the export process competes with RNA processing and degradation pathways, maintaining a delicate balance vital for accurate gene expression. While these mechanisms are generally conserved across eukaryotes, significant differences exist between yeast and higher eukaryotic cells, particularly due to the more genome complexity of the latter. This review delves into the current research on mRNA export in higher eukaryotic cells, focusing on its role in the broader context of gene expression regulation and highlighting how it interacts with other gene expression processes to ensure precise and efficient gene functionality in complex organisms.

The transcription and transportation of mRNA are coupled processes; however, the mechanisms linking these processes remain unclear. Additionally, the significance of this connection in cancer drug development is poorly understood. To address these issues, we investigated the role of CDK12 kinase, which regulates RNA transcription through the phosphorylation of RNA polymerase II (Pol II) and has a repeated serine-arginine dipeptide (RS domain) involved in mRNA transport. Despite the anticipated uniqueness of CDK12 function, the mechanism by which CDK12 bridges and manages mRNA transcription and transport has not been fully analyzed. Our study revealed that CDK12 interacts with NXF1, a key molecule involved in the export of mRNA from the nucleus to the cytosol. Although CDK12 does not phosphorylate NXF1, we found that NXF1 unexpectedly stabilized the CDK12 protein, suggesting that NXF1 mRNA export activity indirectly affects mRNA transcriptional activity by modifying the protein level of CDK12. Furthermore, CDK12 recruited other essential RNA transporters, specifically the exon junction complex (EJC) and THO complexes, into the CDK12-NXF1 axis through its kinase activity. These observations provide insights into the mechanisms linking mRNA transcription and transport through the formation of a novel CDK12-NXF1 complex that involves EJC and THO. Importantly, the expression level of NXF1 influences sensitivity to CDK12 inhibitors, which are emerging as novel anti-cancer drug candidates. This highlights the importance of considering the relationship between mRNA transcription and transport when targeting RNA transcription in cancer therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daisuke Morishita reports a relationship with Chordia Therapeutics Inc. that includes: employment and equity or stocks. Shunsuke Ebara reports a relationship with Chordia Therapeutics Inc. that includes: employment and equity or stocks. Misaki Yoshida reports a relationship with Chordia Therapeutics Inc. that includes: employment. Hiroko Yamakawa reports a relationship with Chordia Therapeutics Inc. that includes: employment and equity or stocks. Kenichiro Shimokawa reports a relationship with Chordia Therapeutics Inc. that includes: employment and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

SUN5 , a testis-specific gene, is associated with acephalic spermatozoa syndrome (ASS). Here, we demonstrate that Sun5 is involved in mRNA export. In Sun5 -knockout mice ( Sun 5 -/- ), poly(A) + RNA accumulates in the nuclei of germ cells, leading to reduced sperm counts, decreased sperm motility and disrupted sperm head-to-tail junctions. Additionally, in the GC-2 germ cell line with RNA interference of Sun5 , heterogeneous nuclear ribonucleoproteins (hnRNPs) and poly (A) + RNA (mainly mRNA) are retained in the nucleus. Further mechanistic studies reveal that Sun5 interacts with Nxf1 (nuclear RNA export factor 1) and nucleoporin 93 (Nup93). Interference with Nup93 inhibits mRNA export. Treatment with leptomycin B to block the CRM1 pathway indicates that Sun5 regulates mRNA export through an Nxf1-dependent pathway. In Sun5 mice, the binding of Nxf1 and Nup93 decreases due to loss of Sun5 function, and the process of submitting Nxf1-binding mRNPs to Nup93 is inhibited, resulting in abnormal spermatogenesis. Together, these data may elucidate a novel pathway for mRNA export in male germ cells.-/- mice, the binding of Nxf1 and Nup93 decreases due to loss of Sun5 function, and the process of submitting Nxf1-binding mRNPs to Nup93 is inhibited, resulting in abnormal spermatogenesis. Together, these data may elucidate a novel pathway for mRNA export in male germ cells.

The relationship between transcription and aging is one that has been studied intensively and experimentally with diverse attempts. However, the impact of the nuclear mRNA export on the aging process following its transcription is still poorly understood, although the nuclear events after transcription are coupled closely with the transcription pathway because the essential factors required for mRNA transport, namely TREX, TREX-2, and nuclear pore complex (NPC), physically and functionally interact with various transcription factors, including the activator/repressor and pre-mRNA processing factors. Dysregulation of the mediating factors for mRNA export from the nucleus generally leads to the aberrant accumulation of nuclear mRNA and further impairment in the vegetative growth and normal lifespan and the pathogenesis of neurodegenerative diseases. The optimal stoichiometry and density of NPC are destroyed during the process of cellular aging, and their damage triggers a defect of function in the nuclear permeability barrier. This review describes recent findings regarding the role of the nuclear mRNA export in cellular aging and age-related neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Human ZC3H11A is an RNA-binding zinc finger protein involved in mRNA export and required for the efficient growth of human nuclear replicating viruses. Its biochemical properties are largely unknown so our goal has been to produce the protein in a pure and stable form suitable for its characterization. This has been challenging since the protein is large (810 amino acids) and with only the N-terminal zinc finger domain (amino acids 1–86) being well structured, the remainder is intrinsically disordered. Our production strategies have encompassed recombinant expression of full-length, truncated and mutated ZC3H11A variants with varying purification tags and fusion proteins in several expression systems, with or without co-expression of chaperones and putative interaction partners. A range of purification schemes have been explored. Initially, only truncated ZC3H11A encompassing the zinc finger domain could successfully be produced in a stable form. It required recombinant expression in insect cells since expression in E. coli gave a protein that aggregated. To reduce problematic nucleic acid contaminations, Cys8, located in one of the zinc fingers, was substituted by Ala and Ser. Interestingly, this did not affect nucleic acid binding, but the full-length protein was stabilised while the truncated version was insoluble. Ultimately, we discovered that when using alkaline buffers (pH 9) for purification, full-length ZC3H11A expressed in Sf9 insect cells was obtained in a stable and >90 % pure form, and as a mixture of monomers, dimers, tetramers and hexamers. Many of the challenges experienced are consistent with its predicted structure and unusual charge distribution. • Methods for production of ZC3H11A suited for biochemical studies have been established. • The production of stable and pure full length ZC3H11A requires purification at alkaline pH. • ZC3H11A stability but not capability of binding nucleic acid is influenced by substitution of Cys-8 by Ala. [ABSTRACT FROM AUTHOR]

The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export. In this review, we will briefly highlight some of these recent findings and discuss how live cell SMI approaches may be used to further our current understanding of mRNA export and gene expression.

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level. [Display omitted] • NUP153 regulates the localization of BRD4 condensates relevant to SEs • The intrinsically disordered regions of NUP153 are responsible for trapping SEs • The tethering of SEs to NUP153 drives TP63 oncogene expression via mRNA export Hazawa et al. demonstrate that protein-protein interaction between the intrinsically disordered regions of NUP153 and the coactivator BRD4 regulates the localization of phase-separated super-enhancer at nuclear periphery. This spatial organization drives TP63 oncogene expression by ensuring effective mRNA export. [ABSTRACT FROM AUTHOR]

Nuclear export of mRNAs is a critical regulatory step in eukaryotic gene expression. The mRNA transcript undergoes extensive processing, and is loaded with a set of RNA-binding proteins (RBPs) to form export-competent messenger ribonucleoprotein particles (mRNPs) in the nucleus. During the transit of mRNPs through the nuclear pore complex (NPC), the DEAD-box ATPase - DDX19 (herein referring to DDX19A and DDX19B) - remodels mRNPs at the cytoplasmic side of the NPC, by removing a subset of RNA-binding proteins to terminate mRNP export. This requires the RNA-dependent ATPase activity of DDX19 and its dynamic interactions with Gle1 and Nup214. However, the regulatory mechanisms underlying these interactions are unclear. We find that DDX19 gets covalently attached with a small ubiquitin-like modifier (SUMO) at lysine 26, which enhances its interaction with Gle1. Furthermore, a SUMOylation-defective mutant of human DDX19B, K26R, failed to provide a complete rescue of the mRNA export defect caused by DDX19 depletion. Collectively, our results suggest that SUMOylation fine-tunes the function of DDX19 in mRNA export by regulating its interaction withGle1. This study identifies SUMOylation of DDX19 as a modulatory mechanism during the mRNA export process. [ABSTRACT FROM AUTHOR]

Abstract Background The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation. Results To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1). Conclusions Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.

The eukaryotic translation initiation factor eIF4E acts as a multifunctional factor that simultaneously influences mRNA processing, export, and translation in many organisms. Its multifactorial effects are derived from its capacity to bind to the methyl-7-guanosine cap on the 5'end of mRNAs and thus can act as a cap chaperone for transcripts in the nucleus and cytoplasm. In this review, we describe the multifactorial roles of eIF4E in major mRNA-processing events including capping, splicing, cleavage and polyadenylation, nuclear export and translation. We discuss the evidence that eIF4E acts at two levels to generate widescale changes to processing, export and ultimately the protein produced. First, eIF4E alters the production of components of the mRNA processing machinery, supporting a widescale reprogramming of multiple mRNA processing events. In this way, eIF4E can modulate mRNA processing without physically interacting with target transcripts. Second, eIF4E also physically interacts with both capped mRNAs and components of the RNA processing or translation machineries. Further, specific mRNAs are sensitive to eIF4E only in particular mRNA processing events. This selectivity is governed by the presence of cis-acting elements within mRNAs known as USER codes that recruit relevant co-factors engaging the appropriate machinery. In all, we describe the molecular bases for eIF4E's multifactorial function and relevant regulatory pathways, discuss the basis for selectivity, present a compendium of ~80 eIF4E-interacting factors which play roles in these activities and provide an overview of the relevance of its functions to its oncogenic potential. Finally, we summarize early-stage clinical studies targeting eIF4E in cancer.

mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron‐containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole‐cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex. [ABSTRACT FROM AUTHOR]

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export. [Display omitted] • SRSF1 activates the FA pathway by binding and stimulating FANCD2 ubiquitination • FANCD2 ubiquitination is crucial for the formation of NXF1-SRSF1 export complex • FANCD2 monoubiquitination is critical for NXF1-mediated mRNA export • SRSF1 cancer mutants fail to bind FANCD2 and NXF1, leading to inefficient mRNA export While the Fanconi anemia pathway has a well-characterized function in DNA crosslink repair, its newly uncovered role in regulating R-loop metabolism has not been fully understood. Olazabal-Herrero et al. reveal that FANCD2 functions in NXF1-SRSF1-mediated mRNA export regulation, preventing R-loop formation and subsequent genomic instability. [ABSTRACT FROM AUTHOR]

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro , where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress. [Display omitted] • Glucose stress and loss of the DEAD-box ATPase Dbp5 lead to nuclear Nab2 condensation • Nab2 condensates form in vitro and in vivo in an RNA-dependent manner • Nab2 multimerization, but not its intrinsically disordered regions, drives condensation • Stress-induced nuclear poly(A) RNA retention is affected by the state of the Nab2 condensate Heinrich et al. report that a stress-induced block in mRNA export leads to the formation of nuclear condensate-like structures that contain both mRNA and the poly(A)-binding protein Nab2. Preventing Nab2 multimerization affects the material properties of the condensates and the degree of nuclear mRNA retention. [ABSTRACT FROM AUTHOR]