RNA Export Research Articles

The deubiquitylase Ubp15 couples transcription to mRNA export.

Nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed, and exported. The role of ubiquitylation in this process is increasingly recognized but, while a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here we identified deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts with both RNA polymerase II and the nuclear pore complex, and its deletion reverts the nuclear export defect of E3 ligase Rsp5 mutants. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.

Emerging molecular functions and novel roles for the DEAD-box protein Dbp5/DDX19 in gene expression.

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

Identification of a new coronavirus-specific RNA export protein complex

Identification of a new coronavirus-specific RNA export protein complex

Impact of Nuclear Export Pathway on Cytoplasmic HIV-1 RNA Transport Mechanism and Distribution.

HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions.IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

Mutations of the Exportin 1 (XPO1) Gene Predict Shorter Time to First Treatment in 1092 Early Stage Chronic Lymphocytic Leukemia Patients. Α Training/Validation Study

Mutations of the Exportin 1 (XPO1) Gene Predict Shorter Time to First Treatment in 1092 Early Stage Chronic Lymphocytic Leukemia Patients. Α Training/Validation Study

Non-Canonical Functions of Splicing Factors in RNA Metabolism

Noncoding RNA (ncRNA), such as microRNA (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) play roles in plant growth, development, and response to environmental cues via diverse manners. Splicing factors are critical for precursor messenger RNAs (pre-mRNAs) splicing and alternative splicing. Strikingly, over the past few years, an increasing number of studies demonstrated that splicing factors couple splicing to other processes, such as ncRNA biogenesis, nuclear retention, and cytoplasmic export of various RNAs, and even translation. Here, we review the current status of the noncanonical functions of splicing factors in the ncRNAs processing, retention and export of RNAs, and translation in plants.

The Regulatory Properties of the Ccr4-Not Complex.

The mammalian Ccr4–Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. In the nucleus, it is involved in the regulation of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, nuclear RNA surveillance, and DNA damage repair. In the cytoplasm, the Ccr4–Not complex plays a central role in mRNA decay and affects protein quality control. Most of our original knowledge of the Ccr4–Not complex is derived, primarily, from studies in yeast. More recent studies have shown that the mammalian complex has a comparable structure and similar properties. In this review, we summarize the evidence for the multiple roles of both the yeast and mammalian Ccr4–Not complexes, highlighting their similarities.

The eukaryotic translation initiation factor eIF4E elevates steady-state m7G capping of coding and noncoding transcripts

Methyl-7-guanosine (m7G) "capping" of coding and some noncoding RNAs is critical for their maturation and subsequent activity. Here, we discovered that eukaryotic translation initiation factor 4E (eIF4E), itself a cap-binding protein, drives the expression of the capping machinery and increased capping efficiency of ∼100 coding and noncoding RNAs. To quantify this, we developed enzymatic (cap quantification; CapQ) and quantitative cap immunoprecipitation (CapIP) methods. The CapQ method has the further advantage that it captures information about capping status independent of the type of 5' cap, i.e., it is not restricted to informing on m7G caps. These methodological advances led to unanticipated revelations: 1) Many RNA populations are inefficiently capped at steady state (∼30 to 50%), and eIF4E overexpression increased this to ∼60 to 100%, depending on the RNA; 2) eIF4E physically associates with noncoding RNAs in the nucleus; and 3) approximately half of eIF4E-capping targets identified are noncoding RNAs. eIF4E's association with noncoding RNAs strongly positions it to act beyond translation. Coding and noncoding capping targets have activities that influence survival, cell morphology, and cell-to-cell interaction. Given that RNA export and translation machineries typically utilize capped RNA substrates, capping regulation provides means to titrate the protein-coding capacity of the transcriptome and, for noncoding RNAs, to regulate their activities. We also discovered a cap sensitivity element (CapSE) which conferred eIF4E-dependent capping sensitivity. Finally, we observed elevated capping for specific RNAs in high-eIF4E leukemia specimens, supporting a role for cap dysregulation in malignancy. In all, levels of capping RNAs can be regulated by eIF4E.

WRN modulates translation by influencing nuclear mRNA export in HeLa cancer cells

BackgroundThe 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.ResultsTo 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).ConclusionsOur 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.

Molecular mechanism underlying selective inhibition of mRNA nuclear export by herpesvirus protein ORF10

Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1-Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1-Nup98, the ORF10-Rae1-Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10-RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1-Nup98 in regulating host mRNA export.

XPO1E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-Cell Lymphoma

Simple SummaryAlmost 25% of patients with either primary mediastinal B-cell lymphoma (PMBL) or classical Hodgkin lymphoma (cHL) possess a recurrent mutation of the XPO1 gene encoding the major nuclear export protein. The aim of our study was to assess the molecular function of the mutant XPO1 protein. Using several cell models (including CRISPR–Cas9 edited cells) and high throughput techniques, we determined that the export capacity of the mutant XPO1 was not altered. However, mutant XPO1 accumulated in the cytoplasm due to its binding to importin β1 (or IPO1). The targeting of XPO1 is largely efficient for fighting haemopathies. The inhibition of IPO1 could open new therapeutic perspectives for B-cell lymphomas.The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>T) resulting in the E571K substitution within the hydrophobic groove of the protein, the site of cargo binding. We investigated the impact of the XPO1E571K mutation using PMBL/cHL cells having various XPO1 statuses and CRISPR–Cas9-edited cells in which the E571K mutation was either introduced or knocked-out. We first confirmed that the mutation was present in both XPO1 mRNA and protein. We observed that the mutation did not modify the export capacity but rather the subcellular localisation of XPO1 itself. In particular, mutant XPO1 bound to importin β1 modified the nuclear export/import dynamics of relevant cargoes.

NCBP3 positively impacts mRNA biogenesis.

The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5′cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein–protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.

It's the Little Things (in Viral RNA).

Chemical modifications of viral RNA are an integral part of the viral life cycle and are present in most classes of viruses. To date, more than 170 RNA modifications have been discovered in all types of cellular RNA. Only a few, however, have been found in viral RNA, and the function of most of these has yet to be elucidated. Those few we have discovered and whose functions we understand have a varied effect on each virus. They facilitate RNA export from the nucleus, aid in viral protein synthesis, recruit host enzymes, and even interact with the host immune machinery. The most common methods for their study are mass spectrometry and antibody assays linked to next-generation sequencing. However, given that the actual amount of modified RNA can be very small, it is important to pair meticulous scientific methodology with the appropriate detection methods and to interpret the results with a grain of salt. Once discovered, RNA modifications enhance our understanding of viruses and present a potential target in combating them. This review provides a summary of the currently known chemical modifications of viral RNA, the effects they have on viral machinery, and the methods used to detect them.

Strength in Diversity: Nuclear Export of Viral RNAs.

The nuclear export of cellular mRNAs is a complex process that requires the orchestrated participation of many proteins that are recruited during the early steps of mRNA synthesis and processing. This strategy allows the cell to guarantee the conformity of the messengers accessing the cytoplasm and the translation machinery. Most transcripts are exported by the exportin dimer Nuclear RNA export factor 1 (NXF1)–NTF2-related export protein 1 (NXT1) and the transcription–export complex 1 (TREX1). Some mRNAs that do not possess all the common messenger characteristics use either variants of the NXF1–NXT1 pathway or CRM1, a different exportin. Viruses whose mRNAs are synthesized in the nucleus (retroviruses, the vast majority of DNA viruses, and influenza viruses) exploit both these cellular export pathways. Viral mRNAs hijack the cellular export machinery via complex secondary structures recognized by cellular export factors and/or viral adapter proteins. This way, the viral transcripts succeed in escaping the host surveillance system and are efficiently exported for translation, allowing the infectious cycle to proceed. This review gives an overview of the cellular mRNA nuclear export mechanisms and presents detailed insights into the most important strategies that viruses use to export the different forms of their RNAs from the nucleus to the cytoplasm.

Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway

Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.

A gateway to the cytosol

Coronavirus Coronaviruses transform host cell membranes into peculiar double-membrane vesicles that have long been thought to accommodate viral genome replication. However, because these compartments appeared to be completely sealed, it has remained unknown how the newly made viral RNA could be exported to the cytosol for translation and packaging into new virions. Wolff et al. used cryo–electron microscopy to identify a molecular pore that spans the double membrane (see the Perspective by Unchwaniwala and Ahlquist). Six copies of a large coronavirus transmembrane protein formed the core of this structure, which may constitute a viral RNA export channel and provide a target for future antiviral interventions. Science , this issue p. [1395][1]; see also p. [1306][2] [1]: /lookup/doi/10.1126/science.abd3629 [2]: /lookup/doi/10.1126/science.abe0322

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

RNA localization in prokaryotes: Where, when, how, and why.

Only recently has it been recognized that the transcriptome of bacteria and archaea can be spatiotemporally regulated. All types of prokaryotic transcripts-rRNAs, tRNAs, mRNAs, and regulatory RNAs-may acquire specific localization and these patterns can be temporally regulated. In some cases bacterial RNAs reside in the vicinity of the transcription site, but in many others, transcripts show distinct localizations to the cytoplasm, the inner membrane, or the pole of rod-shaped species. This localization, which often overlaps with that of the encoded proteins, can be achieved either in a translation-dependent or translation-independent fashion. The latter implies that RNAs carry sequence-level features that determine their final localization with the aid of RNA-targeting factors. Localization of transcripts regulates their posttranscriptional fate by affecting their degradation and processing, translation efficiency, sRNA-mediated regulation, and/or propensity to undergo RNA modifications. By facilitating complex assembly and liquid-liquid phase separation, RNA localization is not only a consequence but also a driver of subcellular spatiotemporal complexity. We foresee that in the coming years the study of RNA localization in prokaryotes will produce important novel insights regarding the fundamental understanding of membrane-less subcellular organization and lead to practical outputs with biotechnological and therapeutic implications. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Abstract 1545: IGF2BP2 over-expression induces epithelial mesenchymal transition & promotes tumor progression in ESCC cells

Abstract Esophageal cancer (EC) is a common form of malignant tumor with high morbidity & mortality worldwide. IGF2BP2/ IMP2 (insulin-like growth factor 2 mRNA binding protein 2) belongs to a large family of RBPs (RNA binding proteins) that coordinates the export, trafficking, precise localization and translation of RNA in cells. Analysis of TCGA data (results from GEPIA, http://gepia.cancer-pku.cn/) showed that IGF2BP2 over-expressed in ESCA and many other types of human cancers. However, whether and how IGF2BP2 affects EC progression is still unclear. In this study, the expression of IGF2BP2 in esophageal squamous cancer cell lines and normal esophageal epithelial cells(Het) were investigated to explore the relationship between IGF2BP2 and epithelial-mesenchymal transition(EMT)in the ESCC. Methods: IGF2BP2 was knocked down in the Kyse150 cells by siRNA while upregulated by infection lentivirus with IGF2BP2 cDNA in the Eca109 cells. Then Western blotting was used to detect the protein expression of IGF2BP2, p38, P-p38, ERK and EMT markers in these cells. Results: In this study, the results showed that the expression of the epithelial marker, E-cadherin protein increased and the mesenchymal markers, N-cadherin, Snail, and Slug protein decreased after IGF2BP2 knockdown in Kyse150 cells. Meanwhile, the EMT features were promoted in the Eca109 cell after IGF2BP2 upregulation, shown as a reduction in epithelial marker E-cadherin with an increase in mesenchymal markers: N-cadherin, Snail, and Slug. Results from Western blotting showed that IGF2BP2 protein expression was independent of p38 and ERK protein levels. Conclusion: These data indicated that IGF2BP2 could promote the process of EMT in ESCC cells and might serve as a new therapeutic target for the treatment of esophageal cancer. Keywords: IGF2BP2; esophageal cancer; EMT Citation Format: Pei Li, Xuan-Yu HU, Xing-Ge Liu, Bei-Bei Sha, Ying DU, Ping CHEN. IGF2BP2 over-expression induces epithelial mesenchymal transition & promotes tumor progression in ESCC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1545.

Nucleocytoplasmic shuttling of Gle1 impacts DDX1 at transcription termination sites.

Gle1 is a nucleocytoplasmic shuttling protein with well-documented cytoplasmic roles as a modulator of ATP-dependent DEAD-box RNA helicases involved in messenger (m)RNA export, translation initiation and termination, and stress granule dynamics. Here, we identify a novel nuclear role for Gle1 during transcription termination. In HeLa cells treated with a peptide that disrupts Gle1 nucleocytoplasmic shuttling, we detected nuclear accumulation of specific mRNAs with elongated 3′-UTR (untranslated region). Enriched mRNAs were nascently transcribed and accumulated in the nucleus due to a change in transcription state and not due to altered nuclear export. Whereas Gle1 shuttling inhibition did not appear to perturb nuclear DDX19 functions, it did result in increased DDX1 nucleoplasmic localization and decreased DDX1 interactions with Gle1 and the pre-mRNA cleavage stimulation factor CstF-64. An increase in nuclear R-loop signal intensity was also observed with diminished Gle1 shuttling, as well as colocalization of Gle1 at R-loops. Taken together, these studies reveal a nuclear role for Gle1 in coordinating DDX1 function in transcription termination complexes.