The Mammalian Ecdysoneless Protein Interacts with RNA Helicase DDX39A To Regulate Nuclear mRNA Export.

The mammalian orthologue of ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of endoplasmic reticulum stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full-length ECD but not an ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publicly available BC mRNA The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data sets. In both data sets, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In the METABRIC data set, ECD overexpression also correlated with poor patient survival in triple-negative breast cancer (TNBC). Furthermore, ECD KD in ErbB2+ BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export and was associated with impairment of oncogenic traits. These findings provide novel mechanistic insight into the physiological and pathological functions of ECD.

EWI-2 controls nucleocytoplasmic shuttling of EGFR signaling molecules and miRNA sorting in exosomes to inhibit prostate cancer cell metastasis.

Early and accurate diagnosis of prostate cancer (PCa) is extremely important, as metastatic PCa remains hard to treat. EWI-2, a member of the Ig protein subfamily, is known to inhibit PCa cell migration. In this study, we found that EWI-2 localized on both the cell membrane and exosomes regulates the distribution of miR-3934-5p between cells and exosomes. Interestingly, we observed that EWI-2 is localized not only on the plasma membrane but also on the nuclear envelope (nuclear membrane), where it regulates the nuclear translocation of signaling molecules and miRNA. Collectively, these functions of EWI-2 found in lipid bilayers appear to regulate PCa cell metastasis through the epidermal growth factor receptor-mitogen-activated protein kinase-extracellular-signal-regulated kinase (EGFR-MAPK-ERK) pathway. Our research provides new insights into the molecular function of EWI-2 on PCa metastasis, and highlights EWI-2 as a potential PCa biomarker.

SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs.

Dietary microRNAs have been shown to be absorbed by mammals and regulate host gene expression, but the absorption mechanism remains unknown. Here, we show that SIDT1 expressed on gastric pit cells in the stomach is required for the absorption of dietary microRNAs. SIDT1-deficient mice show reduced basal levels and impaired dynamic absorption of dietary microRNAs. Notably, we identified the stomach as the primary site for dietary microRNA absorption, which is dramatically attenuated in the stomachs of SIDT1-deficient mice. Mechanistic analyses revealed that the uptake of exogenous microRNAs by gastric pit cells is SIDT1 and low-pH dependent. Furthermore, oral administration of plant-derived miR2911 retards liver fibrosis, and this protective effect was abolished in SIDT1-deficient mice. Our findings reveal a major mechanism underlying the absorption of dietary microRNAs, uncover an unexpected role of the stomach and shed light on developing small RNA therapeutics by oral delivery.

Visualization of Transiently Expressed mRNA in Plants Using MS2.

RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.

Down-regulation of long non-coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats.

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non-coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up-regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down-regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.

Endometrial microRNAs and their aberrant expression patterns.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They play fundamental roles in several biological processes, including cell differentiation and proliferation, embryo development, organ development, and organ metabolism. Besides regulating the physiological processes, miRNAs regulate various pathological conditions such as tumors, metastases, metabolic diseases, and osteoporosis. Although several studies have been performed on miRNAs, only few studies have described the miRNA expression and functions in human reproductive tract tissues. During menstruation, the human endometrium undergoes extensive cyclic morphological and biochemical modifications before embryo implantation. In addition to the ovarian steroid hormones (estrogen and progesterone), endometrial autocrine or paracrine factors and embryo-derived signals play a significant role in endometrial functions. miRNAs are considered key regulators of gene expression in the human endometrium and implantation process, and their aberrant expression levels are associated with the development of various disorders, including tumorigenesis. In this review, we summarize the studies that show the role of miRNAs in regulating the physiological conditions of the endometrium and the implantation process and discuss the aberrant expression of miRNAs in ectopic pregnancy, endometriosis, and endometrial cancer.

Uncoupling of nucleo-cytoplasmic RNA export and localization during stress.

Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.

Translin: A multifunctional protein involved in nucleic acid metabolism.

Translin, a highly conserved, DNA/RNA binding protein, is abundantly expressed in brain, testis and in certain malignancies. It was discovered initially in the quest to find proteins that bind to alternating polypurines-polypyrimidines repeats. It has been implicated to have a role in RNA metabolism (tRNA processing, RNAi, RNA transport, etc.), transcription, DNA damage response, etc. Studies from human, mice, drosophila and yeast have revealed that it forms an octameric ring, which is important for its function. Translin is a cytoplasmic protein, but under genotoxic stress, it migrates into the nucleus, binds to the break point hot spots and therefore, thought to be involved in chromosomal translocation events as well as DNA damage related response. Its structure is known and DNA binding regions, GTP binding region and regions responsible for homotypic and heterotypic interaction are known. It forms a ball like structure with open central channel for accommodating the substrate nucleic acids. Besides this, translin protein binds to 3' and 5' UTR of certain mRNAs and probably regulates their availability for translation. It is also involved in mRNA transport and cell cycle progression. It forms a heteromeric complex with translin associated factor-X (TRAX) to form C3PO complex which is involved in RNA silencing process. Recently, it has been shown that translin is upregulated under starvation conditions in Drosophila and is involved in the integration of sleep and metabolic rate of the flies. Earlier studies classified translin as a DNA repair protein; however subsequent studies showed that it is a multifunctional protein. With this background, in this review we have summarized the translin biochemical activities, cellular function as well as structural properties of this important protein.

Overlapping motifs on the herpes viral proteins ICP27 and ORF57 mediate interactions with the mRNA export adaptors ALYREF and UIF.

The TREX complex mediates the passage of bulk cellular mRNA export to the nuclear export factor TAP/NXF1 via the export adaptors ALYREF or UIF, which appear to act in a redundant manner. TREX complex recruitment to nascent RNA is coupled with 5' capping, splicing and polyadenylation. Therefore to facilitate expression from their intronless genes, herpes viruses have evolved a mechanism to circumvent these cellular controls. Central to this process is a protein from the conserved ICP27 family, which binds viral transcripts and cellular TREX complex components including ALYREF. Here we have identified a novel interaction between HSV-1 ICP27 and an N-terminal domain of UIF in vivo, and used NMR spectroscopy to locate the UIF binding site within an intrinsically disordered region of ICP27. We also characterized the interaction sites of the ICP27 homolog ORF57 from KSHV with UIF and ALYREF using NMR, revealing previously unidentified binding motifs. In both ORF57 and ICP27 the interaction sites for ALYREF and UIF partially overlap, suggestive of mutually exclusive binding. The data provide a map of the binding sites responsible for promoting herpes virus mRNA export, enabling future studies to accurately probe these interactions and reveal the functional consequences for UIF and ALYREF redundancy.

Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex.

We investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.

RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs.

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.

Expression of tomato prosystemin gene in Arabidopsis reveals systemic translocation of its mRNA and confers necrotrophic fungal resistance.

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.

Exosomal MicroRNA Transport from Salivary Mesenchyme Regulates Epithelial Progenitor Expansion during Organogenesis.

Epithelial-mesenchymal interactions involve fundamental communication between tissues during organogenesis and are primarily regulated by growth factors and extracellular matrix. It is unclear whether RNA-containing exosomes are mobile genetic signals regulating epithelial-mesenchymal interactions. Here we identify that exosomes loaded with mesenchyme-specific mature microRNA contribute mobile genetic signals from mesenchyme to epithelium. The mature mesenchymal miR-133b-3p, loaded into exosomes, was transported from mesenchyme to the salivary epithelium, which did not express primary miR-133b-3p. Knockdown of miR-133b-3p in culture decreased endbud morphogenesis, reduced proliferation of epithelial KIT+ progenitors, and increased expression of a target gene, Disco-interacting protein 2 homolog B (Dip2b). DIP2B, which is involved in DNA methylation, was localized with 5-methylcytosine in the prophase nucleus of a subset of KIT+ progenitors during mitosis. In summary, exosomal transport of miR-133b-3p from mesenchyme to epithelium decreases DIP2B, which may function as an epigenetic regulator of genes responsible for KIT+ progenitor expansion during organogenesis.

Integrative analyses reveal a long noncoding RNA-mediated sponge regulatory network in prostate cancer.

Mounting evidence suggests that long noncoding RNAs (lncRNAs) can function as microRNA sponges and compete for microRNA binding to protein-coding transcripts. However, the prevalence, functional significance and targets of lncRNA-mediated sponge regulation of cancer are mostly unknown. Here we identify a lncRNA-mediated sponge regulatory network that affects the expression of many protein-coding prostate cancer driver genes, by integrating analysis of sequence features and gene expression profiles of both lncRNAs and protein-coding genes in tumours. We confirm the tumour-suppressive function of two lncRNAs (TUG1 and CTB-89H12.4) and their regulation of PTEN expression in prostate cancer. Surprisingly, one of the two lncRNAs, TUG1, was previously known for its function in polycomb repressive complex 2 (PRC2)-mediated transcriptional regulation, suggesting its sub-cellular localization-dependent function. Our findings not only suggest an important role of lncRNA-mediated sponge regulation in cancer, but also underscore the critical influence of cytoplasmic localization on the efficacy of a sponge lncRNA.

Import of Fluorescent RNA into Mitochondria of Living Cells.

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.

PbWoxT1 mRNA from pear (Pyrus betulaefolia) undergoes long-distance transport assisted by a polypyrimidine tract binding protein.

Little is known about the mechanisms by which mRNAs are transported over long distances in the phloem between the rootstock and the scion in grafted woody plants. We identified an mRNA in the pear variety 'Du Li' (Pyrus betulaefolia) that was shown to be transportable in the phloem. It contains a WUSCHEL-RELATED HOMEOBOX (WOX) domain and was therefore named Wox Transport 1 (PbWoxT1). A 548-bp fragment of PbWoxT1 is critical in long-distance transport. PbWoxT1 is rich in CUCU polypyrimidine domains and its mRNAs interact with a polypyrimidine tract binding protein, PbPTB3. Furthermore, the expression of PbWoxT1 significantly increased in the stems of wild-type (WT) tobacco grafted onto the rootstocks of PbWoxT1 or PbPTB3 co-overexpressing lines, but this was not the case in WT plants grafted onto PbWoxT1 overexpressing rootstocks, suggesting that PbPTB3 mediates PbWoxT1 mRNA long-distance transport. We provide novel information that adds a new mechanism with which to explain the noncell-autonomous manner of WOX gene function, which enriches our understanding of how WOX genes work in fruit trees and other species.

Depletion of three combined THOC5 mRNA export protein target genes synergistically induces human hepatocellular carcinoma cell death.

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy.

Mutations driving CLL and their evolution in progression and relapse.

Which genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. Here we identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukaemia (CLL) and matched germline DNA samples, 278 of which were collected in a prospective clinical trial. These include previously unrecognized putative cancer drivers (RPS15, IKZF3), and collectively identify RNA processing and export, MYC activity, and MAPK signalling as central pathways involved in CLL. Clonality analysis of this large data set further enabled reconstruction of temporal relationships between driver events. Direct comparison between matched pre-treatment and relapse samples from 59 patients demonstrated highly frequent clonal evolution. Thus, large sequencing data sets of clinically informative samples enable the discovery of novel genes associated with cancer, the network of relationships between the driver events, and their impact on disease relapse and clinical outcome.

Identification of mRNAs that move over long distances using an RNA-Seq analysis of Arabidopsis/Nicotiana benthamiana heterografts.

Phloem is a conductive tissue that allocates nutrients from mature source leaves to sinks such as young developing tissues. Phloem also delivers proteins and RNA species, such as small RNAs and mRNAs. Intensive studies on plant systemic signaling revealed the essential roles of proteins and RNA species. However, many of their functions are still largely unknown, with the roles of transported mRNAs being particularly poorly understood. A major difficulty is the absence of an accurate and comprehensive list of mobile transcripts. In this study, we used a hetero-graft system with Nicotiana benthamiana as the recipient scion and Arabidopsis as the donor stock, to identify transcripts that moved long distances across the graft union. We identified 138 Arabidopsis transcripts as mobile mRNAs, which we collectively termed the mRNA mobilome. Reverse transcription-PCR, quantitative real-time PCR and droplet digital PCR analyses confirmed the mobility. The transcripts included potential signaling factors and, unexpectedly, more general factors. In our investigations, we found no preferred transcript length, no previously known sequence motifs in promoter or transcript sequences and no similarities between the level of the transcripts and that in the source leaves. Grafting experiments regarding the function of ERECTA, an identified transcript, showed that no function of the transcript mobilized. To our knowledge, this is the first report identifying transcripts that move over long distances using a hetero-graft system between different plant taxa.

Here, there, everywhere. mRNA localization in budding yeast.

mRNA localization and localized translation is a common mechanism that contributes to cell polarity and cellular asymmetry. In metazoan, mRNA transport participates in embryonic axis determination and neuronal plasticity. Since the mRNA localization process and its molecular machinery are rather complex in higher eukaryotes, the unicellular yeast Saccharomyces cerevisiae has become an attractive model to study mRNA localization. Although the focus has so far been on the mechanism of ASH1 mRNA transport, it has become evident that mRNA localization also assists in protein sorting to organelles, as well as in polarity establishment and maintenance. A diversity of different pathways has been identified that targets mRNA to their destination site, ranging from motor protein-dependent trafficking of translationally silenced mRNAs to co-translational targeting, in which mRNAs hitch-hike to organelles on ribosomes during nascent polypeptide chain elongation. The presence of these diverse pathways in yeast allows a systemic analysis of the contribution of mRNA localization to the physiology of a cell.