The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

• MeSH
• G-Quadruplexes * MeSH
• DNA, Intergenic chemistry   genetics   MeSH
• Humans MeSH
• Gene Expression Regulation MeSH
• RNA, Viral MeSH
• RNA chemistry   genetics   MeSH
• RNA Transport MeSH
• Epstein-Barr Virus Nuclear Antigens chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Proteomics * MeSH
• RNA MeSH
• RNA Splicing MeSH
• RNA Transport MeSH
• Trypanosoma cruzi * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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

• MeSH
• Active Transport, Cell Nucleus MeSH
• Gene Knockdown Techniques methods   MeSH
• Membrane Transport Proteins metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Nucleocytoplasmic Transport Proteins metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• RNA, Transfer metabolism   MeSH
• Transcriptome MeSH
• RNA Transport MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

• MeSH
• Acetyltransferases genetics   metabolism   MeSH
• HeLa Cells MeSH
• Intracellular Signaling Peptides and Proteins genetics   metabolism   MeSH
• Nuclear Pore Complex Proteins genetics   metabolism   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Neoplasms genetics   MeSH
• Genomic Instability MeSH
• DNA Damage MeSH
• Proto-Oncogene Proteins genetics   metabolism   MeSH
• DNA Replication * MeSH
• RNA Transport MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle. An imbalance in EJC dosage alters mouse neural stem cell (mNSC) division and is linked to human neurodevelopmental disorders. In quiescent mNSC and immortalized human retinal pigment epithelial (RPE1) cells, centrioles form a basal body for ciliogenesis. Here, we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD2. In contrast to BICD2, the localization of NIN transcripts is EJC-dependent. NIN mRNA encodes a core component of centrosomes required for microtubule nucleation and anchoring. We find that EJC down-regulation impairs both pericentriolar material organization and ciliogenesis. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.

• MeSH
• Autoantigens metabolism   MeSH
• Cell Cycle MeSH
• Centrosome metabolism   MeSH
• Cilia metabolism   MeSH
• Cytoskeletal Proteins metabolism   MeSH
• DEAD-box RNA Helicases metabolism   MeSH
• Eukaryotic Initiation Factor-4A metabolism   MeSH
• Exons genetics   MeSH
• Nuclear Proteins metabolism   MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Neural Stem Cells metabolism   MeSH
• Cell Proliferation MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Protein Biosynthesis MeSH
• RNA Transport * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Cell Nucleus metabolism   MeSH
• HeLa Cells MeSH
• RecQ Helicases genetics   MeSH
• Werner Syndrome Helicase metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Metabolic Networks and Pathways physiology   MeSH
• Cell Line, Tumor MeSH
• Neoplasms metabolism   MeSH
• Oxidation-Reduction MeSH
• RNA Processing, Post-Transcriptional physiology   MeSH
• Cell Proliferation physiology   MeSH
• RNA-Binding Proteins metabolism   MeSH
• RNA Transport physiology   MeSH
• Werner Syndrome metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo a complex maturation pathway containing multiple steps in the nucleus and in the cytoplasm. snRNP biogenesis is strictly proofread and several quality control checkpoints are placed along the pathway. Here, we analyzed the fate of small nuclear RNAs (snRNAs) that are unable to acquire a ring of Sm proteins. We showed that snRNAs lacking the Sm ring are unstable and accumulate in P-bodies in an LSm1-dependent manner. We further provide evidence that defective snRNAs without the Sm binding site are uridylated at the 3' end and associate with DIS3L2 3'→5' exoribonuclease and LSm proteins. Finally, inhibition of 5'→3' exoribonuclease XRN1 increases association of ΔSm snRNAs with DIS3L2, which indicates competition and compensation between these two degradation enzymes. Together, we provide evidence that defective snRNAs without the Sm ring are uridylated and degraded by alternative pathways involving either DIS3L2 or LSm proteins and XRN1.

• MeSH
• Exoribonucleases metabolism   MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Organelles metabolism   MeSH
• SMN Complex Proteins metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• RNA, Small Nuclear chemistry   metabolism   MeSH
• Base Sequence MeSH
• RNA Stability MeSH
• RNA Transport * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.

• MeSH
• Arabidopsis drug effects   genetics   microbiology   MeSH
• Botrytis drug effects   physiology   MeSH
• Fluorescence MeSH
• Plants, Genetically Modified MeSH
• Plant Roots drug effects   genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Plant Diseases genetics   microbiology   MeSH
• Disease Resistance drug effects   genetics   MeSH
• Peptides pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Seedlings drug effects   growth & development   physiology   MeSH
• Solanum lycopersicum microbiology   MeSH
• Subcellular Fractions metabolism   MeSH
• RNA Transport drug effects   genetics   MeSH
• Plant Shoots drug effects   genetics   MeSH
• Green Fluorescent Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Cytoplasm genetics   metabolism   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation MeSH
• Nucleoside Q metabolism   MeSH
• Pentosyltransferases genetics   metabolism   MeSH
• RNA, Transfer, Phe genetics   metabolism   MeSH
• RNA, Transfer, Tyr genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• RNA Splicing MeSH
• RNA Transport MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Methods of in vivo visualization and manipulation of mitochondrial genetic machinery are limited due to the need to surpass not only the cytoplasmic membrane but also two mitochondrial membranes. Here, we employ the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mammalian mitochondria, to construct an import system for in vivo targeting of mitochondrial (mt) DNA or mtRNA, in order to provide fluorescence hybridization of the desired sequences.

• MeSH
• Hep G2 Cells MeSH
• Fluorescent Dyes MeSH
• In Situ Hybridization, Fluorescence methods   MeSH
• Humans MeSH
• DNA, Mitochondrial genetics   MeSH
• Mitochondria genetics   MeSH
• Cell Line, Tumor MeSH
• RNA, Ribosomal, 5S genetics   MeSH
• RNA genetics   MeSH
• Transfection methods   MeSH
• RNA Transport genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH