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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.

The passage of mRNAs through the nuclear pores into the cytoplasm is essential in all eukaryotes. For regulation, mRNA export is tightly connected to the full machinery of nuclear mRNA processing, starting at transcription. Export competence of pre-mRNAs gradually increases by both transient and permanent interactions with multiple RNA processing and export factors. mRNA export is best understood in opisthokonts, with limited knowledge in plants and protozoa. Here, I review and compare nuclear mRNA processing and export between opisthokonts and Trypanosoma brucei. The parasite has many unusual features in nuclear mRNA processing, such as polycistronic transcription and trans-splicing. It lacks several nuclear complexes and nuclear-pore-associated proteins that in opisthokonts play major roles in mRNA export. As a consequence, trypanosome mRNA export control is not tight and export can even start co-transcriptionally. Whether trypanosomes regulate mRNA export at all, or whether leakage of immature mRNA to the cytoplasm is kept to a low level by a fast kinetics of mRNA processing remains to be investigated. mRNA export had to be present in the last common ancestor of eukaryotes. Trypanosomes are evolutionary very distant from opisthokonts and a comparison helps understanding the evolution of mRNA export.

The DEAD-box protein (DBP) Dbp5p, 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 [1–9]. Localization of the protein to various cellular compartments (nucleoplasm, nuclear envelope, and cytoplasm) highlights the ability of Dbp5p to modulate different stages of the RNA lifecycle. While Dbp5p has been well studied for >20 years, several critical questions remain regarding the mechanistic principles that govern Dbp5p localization, substrate selection, and functions in gene expression. This review aims to take a holistic view of the proposed functions of Dbp5p and evaluate models that accommodate current published data.

In eukaryotes, the transcription of mRNA takes place in the nucleus of each cell. Already during transcription, various proteins bind the mRNA to prepare it for export from the nucleus so that the information of the genome can be translated into a protein. An essential part of this machinery is the TREX complex, which forms an mRNP together with the mRNA and prepares it for export. This is highly conserved in animals, fungi and plants and consists in Saccharomyces cerevisiae of the heteropentameric THO complex and the RNA helicase Sub2 (UAP56/DDX39 in humans). In addition, other RNA binding proteins (RBPs) such as the RNA annealing protein Yra1 (AlyRef in humans) are recruited. In humans, CIP29/SARNP (in Arabidopsis thaliana MOS11) also interacts with the complex. These RBPs play an important role in mRNA export, but their exact functions are not yet known Sub2 is a conserved DEAD-box RNA helicase from S. cerevisiae and is involved in splicing and mRNA export. While deletion of Sub2 leads to cell death, overexpression rescues the mRNA export defect of a non-functioning THO complex. Although Sub2 has an essential role in mRNA export and splicing, its biochemical activities in these processes are largely unclear. In this work, therefore, the biochemical properties of Sub2 were characterised and its regulation by interaction partners was studied in detail in vitro. Suitable biochemical real-time assays were developed for this purpose. In order to be able to follow important conformational changes in the course of the regulation of Sub2 in the future, the prerequisites for Förster resonance energy transfer (FRET) experiments were created. For the necessary site-specific labelling of Sub2, the existing conserved cysteine residues were replaced and additional cysteine residues were introduced at strategic positions on the surface. The function of the conserved cysteines is currently unclear, but the cysteine-free Sub2 variant does not lead to an obvious phenotype in vivo. First labelling experiments with fluorescent dyes show that the prerequisites for FRET experiments have been created. The starting point for the investigations on the regulation of Sub2 by Tho1 was the fact that the human orthologues UAP56 and CIP29/SARNP form an ATP-dependent complex which leads to the stimulation of ATPase and helicase activity. In this work, ATP-dependent ternary complex formation of Sub2 with Tho1 and RNA was demonstrated. However, Tho1 inhibits the helicase activity of Sub2, which is caused by a previously unknown annealing activity of Tho1. This new activity is evolutionarily conserved and could also be demonstrated for the human CIP29/SARNP, as well as the SARNP/Tho1 orthologue from the thermophilic fungus Chaetomium thermophilum. Furthermore, a detailed mutational analysis of Tho1 experimentally identified the conserved RNA-binding domain as an interaction domain for Sub2. This finding is consistent with the recent prediction of the structures of relevant protein complexes in S. cerevisiae. With the help of further biochemical and cell biological assays, first indications for a new function of Tho1 in mRNA export could also be found. While deletion of Tho1 does not show any phenotype, overexpression of Tho1 (similar to Sub2) rescues several phenotypes (hyperrecombination, transcription and mRNA export defect) of a non-functioning THO complex. The phenotype can be manifested by an accumulation of R-loops and indeed overexpression of Tho1 decreases the number of R-loops in vivo. In vitro experiments show that both Sub2 and, surprisingly, Tho1 alone are already capable of resolving R-loop-like structures. While the resolution of R-loops by Sub2 was expected, as the human orthologue UAP56/DDX39B has already been identified as an important cotranscriptional RNA/DNA helicase, the role of Tho1 is surprising and novel. However, to what extent the interaction of Tho1 and Sub2 is relevant for this function and whether this is evolutionarily conserved is yet to be seen.

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.

The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO–UAP56/DDX39B–ALYREF). TREX selectively binds mRNA maturation marks and licenses mRNA for nuclear export by loading the export factor NXF1–NXT1. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. Here, we report the cryo-electron microscopy structure of the human THO–UAP56/DDX39B complex at 3.3 Å resolution. The seven-subunit THO–UAP56/DDX39B complex multimerizes into a 28-subunit tetrameric assembly, suggesting that selective recognition of mature mRNA is facilitated by the simultaneous sensing of multiple, spatially distant mRNA regions and maturation marks. Two UAP56/DDX39B RNA helicases are juxtaposed at each end of the tetramer, which would allow one bivalent ALYREF protein to bridge adjacent helicases and regulate the TREX–mRNA interaction. Our structural and biochemical results suggest a conserved model for TREX complex function that depends on multivalent interactions between proteins and mRNA., eLife digest The DNA of human and other eukaryotic cells is stored inside a compartment called the nucleus. DNA carries the genetic code and provides a blueprint for all of the cell’s proteins. However, protein production occurs outside the nucleus, in the main body of the cell. To transmit genetic information from one compartment to the other, the DNA sequences are first transcribed into another molecule called messenger RNA, or mRNA for short. Once made, mRNA exits the nucleus and enters the cell’s main body to encounter the machinery that translates its sequence into a protein. Before mRNA can exit the nucleus, it must first undergo a series of modifications, which result in the mRNA molecule being successively bound to specific proteins. Once mRNA has passed through these steps, it is recognized by the transcription-and-export complex, or TREX for short, which is comprised of several proteins. When TREX binds to mRNA, it adds on a final protein which allows the mRNA molecule to be transported out of the nucleus. However, it remained unclear how TREX selects the completed mRNA-protein complexes that are ready for export while at the same time recognizing the wide variety of mRNA molecules produced by cells. Now, Pühringer and Hohmann et al. have identified the first three-dimensional structure of the core of the human TREX complex using a technique called cryo-electron microscopy. This revealed that the seven proteins of the TREX core assemble into a large complex that has four copies of each protein. The structure suggests that TREX can bind to mRNA and its attached proteins in various ways. These different binding arrangements may help the complex select which mRNA molecules are fully modified and ready to be exported. The structure also sheds light on how mutations in this complex can lead to diseases such as Beaulieu–Boycott–Innes syndrome (BBIS). This work will help guide future research into the activity of TREX, including how its structure changes when it binds to mRNA and deposits the final transport protein. Identifying these structures will make it easier to design experiments that target specific aspects of TREX activity and provide new insights into how these complexes work.

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.

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate (IP6) binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel ten amino acid aggregation prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and non-stressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

Significance There is a strong need for the development of new antiviral therapies, and this study sheds light on a host–virus interaction that is significant for a number of medically important human viruses. The study also suggests that the RNA-binding zinc finger CCCH-type containing 11A (ZC3H11A) protein takes part in a mechanism that facilitates nuclear export of mRNA, particularly under cellular stress, a mechanism that has been “hijacked” by several nuclear-replicating viruses to promote their replication. ZC3H11A is therefore a potential target for development of an antiviral therapy., The zinc finger CCCH-type containing 11A (ZC3H11A) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export (TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A–cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication.

Summary In the human genome, most genes undergo splicing, and patterns of codon usage are splicing dependent: guanine and cytosine (GC) content is the highest within single-exon genes and within first exons of multi-exon genes. However, the effects of codon usage on gene expression are typically characterized in unspliced model genes. Here, we measured the effects of splicing on expression in a panel of synonymous reporter genes that varied in nucleotide composition. We found that high GC content increased protein yield, mRNA yield, cytoplasmic mRNA localization, and translation of unspliced reporters. Splicing did not affect the expression of GC-rich variants. However, splicing promoted the expression of AT-rich variants by increasing their steady-state protein and mRNA levels, in part through promoting cytoplasmic localization of mRNA. We propose that splicing promotes the nuclear export of AU-rich mRNAs and that codon- and splicing-dependent effects on expression are under evolutionary pressure in the human genome., Graphical Abstract, Highlights • Codon usage of human protein-coding genes is splicing- and position-dependent • Splicing enhances the expression of genes with low GC content • High GC content increases cytoplasmic mRNA localization • 5′ terminal fusion of GC-rich sequences can be used to enhance expression, Mordstein et al. report an unexpected effect of splicing in human cells, whereby splicing preferentially enhances the expression of genes with low GC content. This might partially explain the high number of introns found in mammalian genomes.

Origin recognition complex (ORC), a heteromeric six-subunit complex, is the central component of the eukaryotic pre-replication complex. Recent data from yeast, frogs, flies and mammals present compelling evidence that ORC and its individual subunits have nonreplicative functions as well. The majority of these functions, such as heterochromatin formation, chromosome condensation, and segregation are dependent on ORC-DNA interactions. Furthermore, ORC is involved in the control of cell division via its participation in centrosome duplication and cytokinesis. Recent findings have also demonstrated a direct interaction between ORC and mRNPs and highlighted an essential role of ORC in mRNA nuclear export. Along with the growth of evolutionary complexity of organisms, ORC complex functions become more elaborate and new functions of the ORC sub-complexes and individual subunits have emerged.

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.

Components of the translation apparatus, including ribosomal proteins, have been found in cell nuclei in various organisms. Components of the translation apparatus are involved in various nuclear processes, particularly those associated with genome integrity control and the nuclear stages of gene expression, such as transcription, mRNA processing, and mRNA export. Components of the translation apparatus control intranuclear trafficking; the nuclear import and export of RNA and proteins; and regulate the activity, stability, and functional recruitment of nuclear proteins. The nuclear translocation of these components is often involved in the cell response to stimulation and stress, in addition to playing critical roles in oncogenesis and viral infection. Many components of the translation apparatus are moonlighting proteins, involved in integral cell stress response and coupling of gene expression subprocesses. Thus, this phenomenon represents a significant interest for both basic and applied molecular biology. Here, we provide an overview of the current data regarding the molecular functions of translation factors and ribosomal proteins in the cell nucleus.

Multiple TREX mRNA export complex subunits (e.g., THOC1, THOC2, THOC5, THOC6, THOC7) have now been implicated in neurodevelopmental disorders (NDDs), neurodegeneration and cancer. We previously implicated missense and splicing-defective THOC2 variants in NDDs and a broad range of other clinical features. Here we report 10 individuals from nine families with rare missense THOC2 variants including the first case of a recurrent variant (p.Arg77Cys), and an additional individual with an intragenic THOC2 microdeletion (Del-Ex37-38). Ex vivo missense variant testing and patient-derived cell line data from current and published studies show 9 of the 14 missense THOC2 variants result in reduced protein stability. The splicing-defective and deletion variants result in a loss of small regions of the C-terminal THOC2 RNA binding domain (RBD). Interestingly, reduced stability of THOC2 variant proteins has a flow-on effect on the stability of the multi-protein TREX complex; specifically on the other NDD-associated THOC subunits. Our current, expanded cohort refines the core phenotype of THOC2 NDDs to language disorder and/or ID, with a variable severity, and disorders of growth. A subset of affected individuals' has severe-profound ID, persistent hypotonia and respiratory abnormalities. Further investigations to elucidate the pathophysiological basis for this severe phenotype are warranted.

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

The influenza A virus replicates in a broad range of avian and mammalian species by hijacking cellular factors and processes. Avian influenza A viruses (AIVs) generally propagated poorly in mammalian cells, but some mutants of virus-encoded RNA polymerase components, especially PB2 subunit, can overcome host restriction. Host factors associated with PB2 may be essential for efficient AIV replication in mammalian cells. Here, we infected human cells with the PB2 Flag-tagged replication-competent recombinant AIV and identified cellular proteins that coprecipitate with PB2 protein by mass spectrometry. We confirmed one of the coprecipitating host factors, DEAD-box protein eIF4A3, that interacts with viral PB2, PB1, and NP proteins. Depletion of endogenous eIF4A3 significantly reduced virus replication. Later studies showed that eIF4A3 is essential for viral RNA polymerase activity and viral RNAs synthesis. Upon systematic dissection of the influenza virus progeny mRNA generation, from pre-mRNA processing to nuclear export, we found that the depletion of eIF4A3 resulted in significant defects in the ratio of M2 to M1 and NS2 to NS1, and the proportion of viral spliced mRNA in the nucleus increased, indicating that eIF4A3 plays a significant function in viral nascent intron mRNA splicing and spliced mRNA (M2 and NS2) nuclear export. Additionally, we confirmed that in specific deletion of eIF4A3, the synthesis of reduced NS2 can significantly impair neo-synthetized viral ribonucleoprotein (vRNP) nuclear export. Taken together, our findings revealed that eIF4A3 is a key mediator of AIV polymerase activity, mRNA splicing, and spliced mRNA nuclear export.

UPF1 is an RNA helicase that is required for efficient nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3'UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from Drosophila, that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and mRNA export from the nucleus.

Summary: The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3′ UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

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.

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

17 páginas, 12 figuras., Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis., PO-C, JS-Q and CN-C were supported by the FPI, FPU and PROMETEO (BES-2012058587, FPU15/03862, PROMETEO 2016/093). This study was supported by funds to SR-N from the Spanish MINECO (BFU2011-23418, BFU2014-57636) and the Generalitat Valenciana (PROMETEO 2012/061 and ACOMP2014/061), and to JEP-O (BFU2016-77728-C3-3-P, PROMETEOII 2015/006).

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.

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in eukaryotic cells, and it facilitates and regulates the bidirectional transfer of molecules between the nucleus and cytoplasm. One of the essential functions of the NPC is to directly regulate the export of mature mRNAs, but the mechanism is not well understood, especially in humans. Export of mRNA is completed at the cytoplasmic side of the NPC, where ATPase activity of the DEAD-box helicase DDX19 is specifically activated by the NPC components Gle1, Nup42, and Nup214. The mRNA export factor Gle1 is an essential nucleoporin, and Gle1 dysfunction has been linked to human diseases. We show that the Gle1-Nup42 interaction is highly conserved with X-ray crystal structures and that the thermostability of Gle1 is highly dependent on Nup42. We also find that disease-linked mutants of Gle1 show strongly altered thermostability. Analysis of DDX19 steady-state ATPase activity reveals a novel mode of activation by Gle1 in humans. Structural studies of DDX19 complement the biochemical characterization of DDX19, and we have proposed a working model for the DDX19 catalytic cycle. An outstanding question remains in this model, as the details of DDX19-mediated removal of the export factor NXF1-NXT1 from mRNA are unknown. We describe a method for large-scale purification of retroviral constitutive transport element (CTE) RNA, which hijacks NXF1-NXT1 to be exported through the NPC, for X-ray crystallographic studies of a NXF1-NXT1-CTE complex. Together, these results provide a detailed structural and functional description of mRNA export regulation by the NPC and a framework for understanding the molecular basis of human disease linked to Gle1 and viral proliferation.

Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and three maternally inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins. We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders, were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such as mRNA export, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in humans. Refereed/Peer-reviewed

Control of gene expression is crucial for parasite survival and is the result of a series of processes that are regulated to permit fine-tuning of gene expression in response to biological changes during the life-cycle of apicomplexan parasites. Control of mRNA nuclear export is a key process in eukaryotic cells but is poorly understood in apicomplexan parasites. Here, we review recent knowledge regarding this process with an emphasis on T. gondii. We describe the presence of divergent orthologs and discuss structural and functional differences in export factors between apicomplexans and other eukaryotic lineages. Undoubtedly, the use of the CRISPR/Cas9 system in high throughput screenings associated with the discovery of mRNA nuclear export complexes by proteomic analysis will contribute to identify these divergent factors. Ligand-based or structure-based strategies may be applied to investigate the potential use of these proteins as targets for new antiprotozoal agents. Electronic supplementary material The online version of this article (10.1186/s13071-018-2648-4) contains supplementary material, which is available to authorized users.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

Nuclear mRNA export is a highly complex and regulated process in cells. Cellular transcripts must undergo successful maturation processes, including splicing, 5'-, and 3'-end processing, which are essential for assembly of an export competent ribonucleoprotein particle. Many viruses replicate in the nucleus of the host cell and require cellular mRNA export factors to efficiently export viral transcripts. However, some viral mRNAs undergo aberrant mRNA processing, thus prompting the viruses to express their own specific mRNA export proteins to facilitate efficient export of viral transcripts and allowing translation in the cytoplasm. This review will focus on the Kaposi’s sarcoma-associated herpesvirus ORF57 protein, a multifunctional protein involved in all stages of viral mRNA processing and that is essential for virus replication. Using the example of ORF57, we will describe cellular bulk mRNA export pathways and highlight their distinct features, before exploring how the virus has evolved to exploit these mechanisms.

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

15 páginas, 11 figuras. El fichero contiene una rectificación en la página 17, The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK, This work was supported by MINECO (Spanish government) Grant BFU2012-33503, the Consolider Ingenio 2010 program (Grant CSD2007-0015), the Fundación Marcelino Botín (to F. P.), and Grant BFU2011-26722 (to E. d. N.). Recipients of an ICREA Acadèmia (Generalitat de Catalunya). Supported by the MINECO Grant BFU2011-23418. Supported by Agence Nationale de la Recherche (Nucleopol and ODynRib-Jeune chercheur program) and Jeune équipe from Fondation pour la Recherche Médicale.

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.

Nucleocytoplasmic transport, the regulated trafficking of macromolecules in and out of the nucleus, occurs primarily through nuclear pore complexes (NPCs). NPCs are massive macromolecular machines embedded in the nuclear envelope which generate ~40 nanometer transport channels to facilitate transport. Because of its size and complexity (~1000 subunits, ~120 MDa), the structure of the NPC has remained poorly understood. This thesis presents a bottom-up approach to understanding the structure and function of the NPC through reconstitution of the proteins and structural and biochemical studies. The first three chapters present work towards determining the composite structure of the symmetric core of the NPC. X-ray crystal structures are described for many of the components of the symmetric core. This includes a heterohexameric coat nucleoporin complex containing Nup120, Nup85, Nup145C, Sec13, Seh1, and Nup84, revealing how these proteins assemble into one of the main subcomplexes in the NPC. Reconstitution of the symmetric core components and analysis of the protein-protein interaction between the components provides a detailed biochemical map for the protein interaction network in the NPC. X-ray crystal structures of overlapping fragments facilitate the generation of accurate atomic model for full-length proteins. An iterative, sequential docking approach is developed to dock these models into a cryoelectron tomographic reconstruction of the human NPC, yielding a composite model for the structure of the symmetric core of the NPC. In the next two chapters, this analysis is extended to the cytoplasmic-specific decorations of the NPC. The structure of the C-terminal domain of Nup358 is reported and its catalytic activity is described. Lastly, reconstitution of human DDX19 activation by the NPC reveals mechanistic insight into how the NPC directly regulates the last step of mRNA export.

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.

Nuclear export of messenger ribonucleic acid (mRNA) is a complex and essential process for a correct gene expression in all eukaryotic cells. The export of mRNA through the nuclear pore complex depends mostly on the crosstalk and coordination of several proteins forming what is known as mRNPs (messenger ribonucleoproteins) that play dynamic, interconnecting roles in the different mRNA biogenesis steps such as pre-mRNA processing, stability, and export.One key protein in this process is Mex67, conserved from yeast to humans, is the major messenger RNA exporter also involved in ribosomal RNA export. Mex67 interacts with Mtr2 to form an evolutionary conserved heterodimer essential for proper mRNA export and subsequently the survival of the cell. Mex67 have been studied for many years, however due to the complexity and interconnectivity of the different processes in mRNA biogenesis, there is yet to uncover many details on the dynamics of the process and the crosstalk between Mex67 and its many partners. In this study, using a combination of biochemical, biophysical, and structural analysis, we characterize the interaction between Mex67 and a novel partner protein called Mip6 (Mex67 interacting protein 6). We were able to reconstitute a stable complex in vitro, and extensively study the mechanism in which the two proteins interact. We also solved the crystal structure of the C-terminal region of Mex67 that interacts with Mip6 and identified the UBA domain of Mex67, known to bind FG nucleoporins and Hpr1 protein as also the site where Mip6 binds. However, little was known about the structure or function of Mip6 and its paralogue Pes4. Here we proved that Mip6 is an RNA binding protein with four RNA recognition motifs that binds RNA in vitro with high affinity. Additionally, its fourth RNA recognition motif was also the site of binding of Mex67. Furthermore, we showed that the Mex67 complex formation with Mip6 RRM4 compromises its ability to bind RNA or vice versa. We also designed a point mutation on Mip6 RRM4 that disrupts its interaction with Mex67 but not with RNA. Subsequent in vivo yeast assays led us to hypothesize a role of Mip6 as an adaptor protein for Mex67 in nuclear export especially upon stress. Additional function of Mip6 was the localization of its bound mRNA to cytoplasmic stress granules in cellular stress conditions.Moreover, the crystal structures of Mip6 RRM3, Pes4 RRM3, Pes4 RRM4, and Pes4 RRM3/4 were also solved. All RRMs adopted a canonical RRM fold with conserved RNP1 and RNP2 sequences normally involved in RNA binding, except Mip6 RRM3 that was missing the aromatic ring in RNP2. In the structure of RNA-free Pes4 RRM3/4, the tandem RRM domains were connected with a flexible disordered linker and no inter-domain contact between them. Finally, although Pes4 RRM4 was binding RNA in vitro, it did not have the ability to interact with Mex67 thus suggesting a separate evolutionary function for Mip6 and Pes4., La exportación nuclear de ácido ribonucleico mensajero (ARNm) es un proceso complejo y esencial para una expresión correcta de los genes en todas las células eucariotas. La exportación de ARNm a través del complejo del poro nuclear depende principalmente de la interacción y coordinación de varias proteínas, que forman lo que se conoce como mRNPs (ribonucleoproteínas mensajeras), que tienen un papel dinámico e interconectado en las diferentes etapas de la biogénesis de ARNm, tales como el procesamiento del pre-ARNm, estabilidad, y exportación.Una proteína clave en este proceso es Mex67, conservada de levaduras a humanos, que es la principal exportadora de ARN mensajero y también está implicada en la exportación de ARN ribosomal. Mex67 interacciona con Mtr2 para formar un heterodímero conservado evolutivamente esencial para una exportación adecuada de ARNm y la consiguiente supervivencia de la célula. Se ha estudiado Mex67 durante muchos años, sin embargo, debido a la complejidad e interconectividad de los diferentes procesos de biogénesis de ARNm, todavía quedan por descubrir muchos detalles de la dinámica del proceso y las interacciones entre Mex67 y sus muchas proteínas asociadas.En este estudio, combinando un análisis bioquímico, biofísico y estructural, hemos caracterizado la interacción entre Mex67 y una nueva proteína asociada denominada Mip6 (proteína 6 que interacciona con Mex67). Hemos podido reconstituir un complejo estable in vitro y estudiar extensivamente el mecanismo por el cual interaccionan estas dos proteínas. También hemos resuelto la estructura cristalográfica de la región C-terminal de Mex67 que interacciona con Mip6 e identificado el dominio UBA de Mex67, conocido por unirse a nucleoporinas FG y a la proteína Hpr1, así como el sitio por el que se une Mip6. No obstante, se sabía muy poco sobre la estructura o la función de Mip6 y su parálogo Pes4. Hemos probado que Mip6 es una proteína de unión a ARN con cuatro motivos de reconocimiento de ARN que se unen a ARN in vitro con una afinidad alta. Además, su cuarto motivo de reconocimiento de ARN es también el sitio de unión a Mex67. Posteriormente, demostramos que la formación del complejo de Mex67 con el dominio RRM4 de Mip6 compromete su capacidad para unir ARN o viceversa. También diseñamos una mutación puntual en el RRM4 de Mip6 que rompe la interacción con Mex67 pero no con el ARN. Los ensayos posteriores in vivo en levaduras nos permitieron establecer una hipótesis sobre el papel de Mip6 como proteína adaptadora para Mex67 en la exportación nuclear, especialmente en condiciones de estrés. Una función adicional de Mip6 era la localización del ARNm que se unía a ella en gránulos de estrés en condiciones de estrés celular.Además, hemos resuelto las estructuras cristalográficas del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 y los RRM3 y 4 de Pes4. Todos los RRMs adoptaron una conformación canónica RRM con secuencias RNP1 y RNP2 conservadas generalmente implicadas en la unión a ARN, excepto el RRM3 de Mip6 que carecía del anillo aromático en RNP2. En la estructura sin ARN de los RRM3 y 4 de Pes4, los dominios RRM tándem estaban conectados por una región flexible desordenada y no había un contacto inter-dominio entre ellos. Finalmente, aunque el RRM4 de Pes4 se unía a ARN in vitro, no presentaba la capacidad de interaccionar con Mex67 lo cual sugiere una divergencia evolutiva de la función de Mip6 y Pes4., L¿exportació nuclear d¿àcid ribonucleic missatger (mRNA) es un procés complex i essencial per a una correcta expresió gènica en totes cèl¿lules eucariotes. L¿exportació del mRNA a través del complex del porus nuclear depén principalment de la interacció i coordinació de diverses proteïnes, que formen el que es coneix com mRNPs (ribonucleoproteïnes missatgeres), que tenen un paper dinàmic i interconnectat en les diferents etapes de la biogènesi d¿ARNm, com el processament del pre-ARNm, estabilitat, localització i exportació.Una proteïna clau en aquest procés és MEX67, conservada de llevats fins a humans, que és la principal exportadora de ARN missatger i també està implicada en l¿exportació de ARN ribosomal. Mex67 interacciona amb Mtr2 per a formar un heterodímer conservat evolutivament essencial per a una exportació adequada d¿ARNm i la consegüent supervivència de la cèl¿lula. S¿ha estudiat Mex67 durant molts anys, però degut a la complexitat i interconectivitat dels diferents processos de biogènesi d¿ARNm, encara queden per descobrir molts detalls de la dinàmica del procés i les interaccions entre Mex67 i les seues moltes proteïnes associades.En aquest estudi, combinant l¿anàlisi bioquímic, biofísic i estructural, hem caracteritzat la interacció entre Mex67 i una nova proteïna associada anomenada Mip6 (proteïna 6 que interacciona amb Mex67). Hem pogut reconstituir un complex estable in vitro i estudiar extensivament el mecanisme pel qual interaccionen estes dos proteïnes. També hem resolt l¿estructura cristal¿logràfica de la regió C-terminal de Mex67 que interacciona amb Mip6 i identificat el domini UBA de Mex67, conegut per unir-se a nucleoporines FG i a la proteïna Hpr1, així com ser el lloc pel que s¿uneix Mip6. No obstant, se sabia molt poc sobre l¿estructura o la funció de Mip6 i el seu paràleg Pes4. Hem comprobat que Mip6 es una proteïna d¿unió a ARN amb quatre motius de reconeixement d¿ARN que s¿uneixen a ARN in vitro amb una afinitat alta. A més, el seu quart motiu de reconeixement d¿ARN és també el lloc d¿unió a Mex67. Posteriorment, demostràrem que la formació del complex de Mex67 amb el domini RRM4 de Mip6 compromet la seua capacitat per a unir ARN o viceversa. També vam dissenyar una mutació puntual en el RRM4 de Mip6 que trenca la interacció amb Mex67 però no amb l¿ARN. Els assajos posteriors in vivo en llevats ens van permetre establir una hipòtesi sobre el paper de Mip6 com a proteïna adaptadora per a Mex67 en l¿exportació nuclear, especialment en condicions d¿estrès. Una funció adicional de Mip6 era la localització de l¿ARNm que s¿unia a ella en grànuls d¿estrès en condicions d¿estrès cel¿lular.A més, hem resolt les estructures cristal¿logràfiques del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 i els RRM3 i 4 de Pes4. Tots els RRMs adoptaren una conformació canònica RRM amb seqüències RNP1 i RNP2 conservades generalment implicades en la unió a ARN, excepte el RRM3 de Mip6 que mancava del anell aromàtic en RNP2. En la estructura sense ARN dels RRM3 i 4 de Pes4, els dominis RRM tàndem estàven conectats per una regió flexible desordenada i no hi havia un contacte interdomini entre ells. Finalment, encara que el RRM4 de Pes4 es unia a ARN in vitro, no presentava la capacitat d¿interaccionar amb Mex67, la cual cosa sugerix una divergencia evolutiva de la funció de Mip6 y Pes4., Mohamad, N. (2017). The RNA binding protein Mip6, a novel cellular partner of Mex67 export factor with implications in mRNA export [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/90397, TESIS

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 compositionallyidentical 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 ondistal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side ofthe NPC.

The eukaryotic translation initiation factor eIF4E plays important roles in controlling the composition of the proteome. Indeed, dysregulation of eIF4E is associated with poor prognosis cancers. The traditional view has been that eIF4E acts solely in translation. However, over the last ∼25 years, eIF4E was found in the nucleus where it acts in mRNA export and in the last ∼10 years, eIF4E was found in cytoplasmic processing bodies (P-bodies) where it functions in mRNA sequestration and stability. The common biochemical thread for these activities is the ability of eIF4E to bind the 7-methylguanosine cap on the 5′ end of mRNAs. Recently, the possibility that eIF4E directly binds some mRNA elements independently of the cap has also been raised. Importantly, the effects of eIF4E are not genome-wide with a subset of transcripts targeted depending on the presence of specific mRNA elements and context-dependent regulatory factors. Indeed, eIF4E governs RNA regulons through co-regulating the expression of groups of transcripts acting in the same biochemical pathways. In addition, studies over the past ∼15 years indicate that there are multiple strategies that regulatory factors employ to modulate eIF4E activities in context-dependent manners. This perspective focuses on these new findings and incorporates them into a broader model for eIF4E function.

In nucleus, eIF4E regulates the nucleus export of specific mRNA. In this study, altered 4E-BP3 (eIF4E-binding protein 3) expression resulted in profoundly affected cyclin D1 protein levels, partially due to changes in the cytoplasmic cyclin D1 mRNA levels in both U2OS and MCF7 cells, whereas altered 4E-BP1 expression did not affect eIF4E-mediated cyclin D1 mRNA export. 4E-BP3 also affected a subset of growth promoting mRNAs exported in an eIF4-dependent manner. Furthermore, 4E-BP3 interacted with dephosphorylated RPA2 (replication protein A2). The results indicated 4E-BP3 acts as an inhibitor of eIF4E-mediated mRNA export in the examined cells, and 4E-BP3 inhibition of eIF4E-mediated mRNA export is regulated by the phosphorylation state of RPA2.Structured summary of protein interactions4EBP3 physically interacts with RPA2 by anti bait coimmunoprecipitation (View interaction)

International audience; The production of mature and export competent mRNP (mRNA ribonucleoprotein) complexes depends on a series of highly coordinated processing reactions. RNA polymerase II (RNAPII) plays a central role in this process by mediating the sequential recruitment of mRNA maturation and export factors to transcribing genes, thereby establishing a strong functional link between transcription and export through nuclear pore complexes (NPC). Growing evidence indicates that post-translational modifications participate in the dynamic association of processing and export factors with mRNAs ensuring that the transitions and rearrangements undergone by the mRNP occur at the right time and place. This review mainly focuses on the role of ubiquitin conjugation in controlling mRNP assembly and quality control from transcription down to export through the NPC. It emphasizes the central role of ubiquitylation in organizing the chronology of events along this highly dynamic pathway. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

The Saccharomyces cerevisiae mRNA export adaptor Yra1 binds the Pcf11 subunit of cleavage-polyadenylation factor CF1A that links export to 3' end formation. We found that an unexpected consequence of this interaction is that Yra1 influences cleavage-polyadenylation. Yra1 competes with the CF1A subunit Clp1 for binding to Pcf11, and excess Yra1 inhibits 3' processing in vitro. Release of Yra1 at the 3' ends of genes coincides with recruitment of Clp1, and depletion of Yra1 enhances Clp1 recruitment within some genes. These results suggest that CF1A is not necessarily recruited as a complete unit; instead, Clp1 can be incorporated co-transcriptionally in a process regulated by Yra1. Yra1 depletion causes widespread changes in poly(A) site choice, particularly at sites where the efficiency element is divergently positioned. We propose that one way Yra1 modulates cleavage-polyadenylation is by influencing co-transcriptional assembly of the CF1A 3' processing factor.

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

The nucleolus is a multifunctional compartment of the eukaryotic nucleus. Besides its well-recognised role in transcription and processing of ribosomal RNA and the assembly of ribosomal subunits, the nucleolus has functions in the processing and assembly of a variety of RNPs and is involved in cell cycle control and senescence and as a sensor of stress. Historically, nucleoli have been tenuously linked to the biogenesis and, in particular, export of mRNAs in yeast and mammalian cells. Recently, data from plants have extended the functions in which the plant nucleolus is involved to include transcriptional gene silencing as well as mRNA surveillance and nonsense-mediated decay, and mRNA export. The nucleolus in plants may therefore have important roles in the biogenesis and quality control of mRNAs.

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., E.V.W. is supported by National Science Foundation Graduate Research Fellowship No. DGE-1122492 and J.V. is supported by an Alberta Innovates Health Solutions Postdoctoral Fellowship. M.M. and Y.M. were supported by a Senior Research Fellowship from the Wellcome Trust (101908/Z/13/Z) and by National Institutes of Health grant R01 GM102869. Research support for B.M. was provided by the Natural Sciences and Engineering Research Council of Canada (RGPIN 435380), Canada Foundation for Innovation (31271), Government of Alberta Research Capacity Program, and Canada Research Chairs program., This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.jmb.2015.12.018

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics.

Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four–amino acid segment, whereas hGle1B has a COOH-terminal 43–amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP–hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39–amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide–treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

Arabidopsis Nup160 and Seh1, encoding two predicted nucleoporins of the Nup107-160 nuclear pore sub-complex, were identified in a reverse genetics screen based on their requirement for basal disease resistance. Both genes also contribute to immunity conferred by Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R proteins and constitutive resistance activated in the deregulated TNL mutant, snc1. Protein amounts of EDS1, a central regulator of TNL-triggered resistance, are reduced in seh1 and severely depleted in nup160 single mutants. Here, we investigate the impact of mutations in Nup160, Seh1 and a third complex member, MOS3/Nup96, on EDS1 protein accumulation in the snc1 auto-immune mutant background. In addition, we examine the subcellular localization of Seh1 in root tissues.

Enfermer le porteur de l’information génétique dans le noyau a obligée la cellule a créé un système de transport complexe, qui permet l’export d’un ARNm du noyau au cytoplasme. Le mécanisme général de l’export des ARNm est encore mal connu, même si les facteurs principaux ont été découverts il y a longtemps. De récents progrès en microscopie nous ont permis d’étudier directement le comportement des ARNm durant le processus d’export. Durant ma maitrise, nous avons été capables de localiser et suivre des ARNm en temps réel pour la première fois chez Saccharomyces cerevisiae. Nous avons créé un gène rapporteur en mettant le gène GLT1 sous le contrôle du promoteur GAL1. Nous avons aussi marqué l’ARNm de GLT1 avec plusieurs boucles PP7. L’ARNm sera visible après l’attachement de plusieurs protéines PP7-GFP aux boucles. En utilisant la technique d’imagerie en cellules vivantes, nous sommes capable de visualiser et suivre chaque ARNm, depuis son relâchement du site de transcription jusqu’à l’export. Une fois relâché du site de transcription, l’ARNm diffuse librement dans le nucléoplasme, mais une fois à la périphérie nucléaire, il commence à « scanner » l’enveloppe nucléaire avant d’être exporté. Nous avons trouvé que le « scanning » dépend de la présence des Myosin Like Proteins (Mlp1p et Mlp2p), protéines qui forment le panier nucléaire, car suite à la délétion de MLP1 et MLP2, les ARNm n’étaient plus capable de « scanner ». Nous avons également trouvé que la partie C-terminale de Mlp1p était nécessaire au « scanning ». De plus, suite à la délétion du gène TOM1, gène codant pour une ubiquitine ligase, les ARNm ont un comportement similaire aux ARNm d’une souche ∆mlp1/mlp2, suggérant que le « scanning » permet à Tom1p d’ubiquitiner Yra1p, ce qui causera son relâchement de l’ARNm. Également, nous avons montré que les ARNm endogènes MDN1 et CBL2 scannent aussi la périphérie nucléaire. Ensemble, nos résultats suggèrent que le scanning est un processus par lequel passent tout les ARNm nucléaire lorsqu’ils se retrouvent à la périphérie du noyau, pour initier plusieurs étapes de réarrangements nécessaires à leurs export. De plus, nous avons examiné le rôle de Yhr127p, une protéine nouvellement identifiée qui se lie à l’ARN. Après avoir marqué cette protéine avec la GFP, nous avons montré qu’elle forme des foci dans le noyau et que ces derniers vont disparaitre suite à l’arrêt de la transcription. La délétion de YHR127 à conduit à une augmentation de la transcription de quelques gènes spécifiques, mais n’affecte pas la capacité de la cellule à exporter les ARNm. Nos résultats suggèrent que cette protéine joue un rôle dans la régulation de la transcription et/ou dans la stabilité de l’ARNm., In eukaryotic cells, the processes of RNA and protein synthesis are spatially separated into two distinct compartments. With this division, a complex pathway of nucleocytoplasmic RNA export has evolved, which to date remains poorly understood. Recent advances in single-molecule microscopy have enabled direct studies focused on investigating the dynamics and kinetics of RNA export. In this Master thesis, we present the first real time visualization of mRNA export in the yeast Saccharomyces cerevisiae. We first generated a GLT1 reporter under the control of the inducible GAL1 promoter, in which the GLT1 mRNA was tagged with an array of PP7 repeats and detected by exogenous PP7-GFP binding protein. Using a single-molecule live cell imaging approach, we were able to visualize and track the behavior of individual mRNAs from the site of transcription to the point of export. Interestingly, we found that once released from the transcription site, single mRNAs diffuse freely in the nucleoplasm, but once they reach the nuclear periphery, they scan the periphery before being exported to the cytoplasm. This scanning behavior was dependent on Myosin Like Proteins (Mlp1p and Mlp2p), which form the basket of the Nuclear Pore Complex (NPC), as mRNAs were not retained at the periphery and were rapidly released into the nucleoplasm in mlp1p/mlp2p double mutant cells. Specifically, we found that the C-terminal part of Mlp1p was important for scanning. Furthermore, mRNAs from cells depleted of the E3 ubiquitin ligase TOM1 had a similar phenotype to mRNAs in mlp1p/mlp2p double mutant cells, suggesting a role for scanning in the Tom1p-mediated release of Yra1p from the RNA. Lastly, we confirmed that endogenous MDN1 and CBL2 mRNAs also exhibit scanning behaviour. Taken together, our results suggest that mRNAs scanning the nuclear periphery is a general behaviour for all mRNAs to initiate the mRNA export process, allowing mRNP arrangement required for export to occur at the nuclear periphery. In addition, we investigated the role of YHR127, a newly identified RNA binding protein, in RNA biogenesis. Notably, we show that GFP-tagged YHR127p formed distinct foci in the nucleus, which were lost upon transcription arrest. Deletion of YHR127 led to an increase in transcript levels of specific genes, but not to a global accumulation of mRNAs in the nucleus, suggesting a role for this protein in regulating transcription and/or mRNA stability.

The human adenovirus (HAdV) early protein E1B55K interacts with E4orf6 to form an E3 ubiquitin ligase complex, which plays key roles in virus replication. To illustrate the reason for the fastidiousness of HAdV-41 in 293 cells, interaction between heterotypic E1B55K and E4orf6 proteins was investigated. HAdV-5 E1B55K could interact with HAdV-41 E4orf6, and vice versa. To form E1B55K/E4orf6 E3 ubiquitin ligase, HAdV-41 E4orf6 recruited Cul2 while HAdV-5 E4orf6 interacted with Cul5. The ligase complex formed by HAdV-5 E1B55K and HAdV-41 E4orf6 could cause the degradation of p53 and Mre11. However, in E1-deleted HAdV-41-infected 293TE7 cells, which expressed HAdV-41 E1B55K, viral late mRNAs were exported from nucleus more efficiently and accumulated to a higher concentration in cytoplasm when compared with that in infected 293 cells. These results suggested that interaction between homotypic E1B55K and E4orf6 was indispensable for efficient export of viral late mRNAs.

To study nucleocytoplasmic transport during multicellular development, we developed a sensitive nuclear protein import assay in living blastoderm embryos. We show that dominant negative truncations of the human nuclear transport receptor karyopherinbeta/Importinbeta (DNImpbeta) disrupt mRNA export and protein import in Drosophila. To test the sensitivity of different developmental processes to nuclear trafficking perturbations, we expressed DNImpbeta behind the morphogenetic furrow of the eye disc, at a time when photoreceptors are patterned and project their axons to the brain. DNImpbeta expression does not disrupt the correct specification of different photoreceptors, but causes a defect in cell adhesion that leads to some photoreceptors descending below the layer of ommatidia. The photoreceptors initially project their axons correctly to the posterior, but later their axons are unable to enter the optic stalk en route to the brain and continue to project an extensive network of misguided axons. The axon guidance and cell adhesion defects are both due to a disruption in the function of Ketel, the Drosophila ortholog of Importinbeta. We conclude that cell adhesion and axon guidance in the eye have specific requirements for nucleocytoplasmic transport, despite involving processes that occur primarily at the cell surface.

Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP). Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems. Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions. Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm.