Point Mutations in the M Domain of PCID2 Impair Its Function in mRNA Export in Drosophila melanogaster.

The PCID2 protein is a component of the eukaryotic TREX-2 complex, which is responsible for mRNA export from the nucleus into the cytoplasm. We have previously shown that Drosophila melanogaster PCID2 is involved in specific mRNA recognition and identified the key amino acids responsible for its interaction with the ras2 RNA. In this work, point mutations of the amino acids were shown to disrupt the PCID2 interaction with cell RNAs and to distort the export of polyA-containing mRNAs from the nucleus into the cytoplasm in Drosophila cells.

SUN5, a testis-specific nuclear membrane protein, participates in recruitment and export of nuclear mRNA in spermatogenesis.

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 ( Sun5 -/-), 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.

Production of stable and pure ZC3H11A - An extensively disordered RNA binding protein.

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.

eIF4E orchestrates mRNA processing, RNA export and translation to modify specific protein production.

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.

Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

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.

HnRNPA2B1 ISGylation Regulates m6A-Tagged mRNA Selective Export via ALYREF/NXF1 Complex to Foster Breast Cancer Development.

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.

Arabidopsis mRNA export factor MOS11: molecular interactions and role in abiotic stress responses.

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.

Replicative aging in yeast involves dynamic intron retention patterns associated with mRNA processing/export and protein ubiquitination.

Saccharomyces cerevisiae (baker's yeast) has yielded relevant insights 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. Research dedicated to unraveling the underlying cellular mechanisms can support 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 utilizing 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 transcriptional program of yeast aging and, more generally, expands the knowledge on the occurrence of alternative splicing in baker's yeast.

TREX-2-ORC Complex of D. melanogaster Participates in Nuclear Export of Histone mRNA.

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.

The FANCI/FANCD2 complex links DNA damage response to R-loop regulation through SRSF1-mediated mRNA export.

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.

Gle1 is required for tRNA to stimulate Dbp5 ATPase activity in vitro and promote Dbp5-mediated tRNA export in vivo in Saccharomyces cerevisiae.

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.

Super-enhancer trapping by the nuclear pore via intrinsically disordered regions of proteins in squamous cell carcinoma cells.

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.

The eukaryotic translation initiation factor eIF4E unexpectedly acts in splicing thereby coupling mRNA processing with translation: eIF4E induces widescale splicing reprogramming providing system-wide connectivity between splicing, nuclear mRNA export and translation.

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.

Glucose stress causes mRNA retention in nuclear Nab2 condensates.

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.

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

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

Interaction of mRNA with the C-Terminal Domain of PCID2, a Subunit of the TREX-2 Complex, Is Required for Its Export from the Nucleus to the Cytoplasm in Drosophila melanogaster.

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.

The Human TREX-2 Complex Interacts with Subunits of the ORC Complex.

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.

Single-molecule quantitation of RNA-binding protein occupancy and stoichiometry defines a role for Yra1 (Aly/REF) in nuclear mRNP organization.

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.

Analysis of the mRNA export protein ZC3H11A in HCMV infection and pan-cancer.

Background: We have previously reported that human cytomegalovirus (HCMV) infection could promote the progression of glioma. Here we discovered a stress-induced nuclear protein ZC3H11A (ZC3) through high-throughput sequencing after HCMV infection, which has been reported recently by our research group in regulating mRNA export under stress conditions. And also, a thorough analysis of ZC3 in pan-cancer and the omics data of ZC3 are yet to be conducted. Methods: The transcriptomes of glioma cells after HCMV infection were assessed by RNA sequencing. ZC3 mRNA and protein level following HCMV infection were validated and measured by qRT-PCR and Western-blot. The RNA sequencing and protein expression information of ZC3 across pan-cancer were analyzed and visualized by R packages. The localization of ZC3 protein was assessed by IHC images from HPA. The ZC3 proteomics and transcriptomics data in different cancers were extracted through the CPTAC data portal, and comparisons were conducted with a Python script. The genetic alteration, survival prognosis, immune infiltration analysis of ZC3 in pan-cancer were analyzed by cBioPortal, TCGA, and TIMER2 databases. The protein interaction networks were revealed by STRING, GEPIA2 and TCGA. Results: Genes in mRNA processing pathways were upregulated after HCMV infection and ZC3 expression in mRNA and protein level was validated. We also discovered that the status of ZC3 were generally at high levels in cancers, although varied among different cancer types. ZC3 protein in tumor cells localized to the nuclear whereas in normal cells it was mainly found in cytoplasmic/membranous. However, from ZC3 proteomics and transcriptomics data in some cancer types, the increase in ZC3 protein was not accompanied by a significant elevation in mRNA level. Additionally, our analysis indicated that elevated ZC3 expression was primarily linked to a negative prognosis in majority cancers but still varied depending on the cancer types. Our annotation analysis suggested that ZC3-related proteins are mainly involved in mRNA processing clusters. Conclusion: We demonstrated that ZC3 significantly impacted by HCMV infection in gliomas. Furthermore, we identified a set of genes exhibiting analogous expression patterns to ZC3H11A in TCGA pan-cancer cohorts, implying a potential functional role for ZC3H11A in mRNA processing. Our study provided valuable insights into the role of a new mRNA export protein ZC3 in HCMV infection and pan-cancer progression. These results lay the foundation for our next research on the regulatory mechanism of ZC3 in virus-infected tumors.

The Cajal body protein p80-coilin forms a complex with the adenovirus L4-22K protein and facilitates the nuclear export of adenovirus mRNA.

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.