Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes1. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation2,3. In C. elegans, these inherited small RNAs have poly (UG) tails4, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.

Main conclusion: The transcripts of transgenic prosystemin (PS) gene are mobile and the PS mRNA can be translated into protein in tomato and tobacco plants. Systemin (SYS) and its precursor protein, prosystemin (PS), are upstream components of the wound-induced signaling pathway in tomato. Although the mobile signal(s) for wound responses has been the subject of considerable research, its identity remains controversial. Intensive studies have revealed the essential role of mRNA on plant systemic signaling. We hypothesize that PS mRNA can act as a transmissible signal in tomato. Herein, we demonstrated that transgenic PS mRNA occurs in leaves located at considerable distances from the initial site of its generation by a transient Agrobacterium-infiltration assay system. We also showed that PS protein is present in the vascular bundle of the distant leaves. Our results indicate that transgenic PS mRNA may be functional as a long-distance signal to modulate systemic defense responses in tomato, providing novel insights into the multifaceted systems by which SYS signaling transports.

Telomerase RNA has long been considered to be a noncoding component of telomerase. However, the expression of the telomerase RNA gene is not always associated with telomerase activity. The existence of distinct TERC gene expression products possessing different functions were demonstrated recently. During biogenesis, hTR is processed by distinct pathways and localized in different cell compartments, depending on whether it functions as a telomerase complex component or facilitates antistress activities as a noncoding RNA, in which case it is either processed in the mitochondria or translated. In order to identify the factors responsible for the appearance and localization of the exact isoform of hTR, we investigated the roles of the factors regulating transcription DSIF (Spt5) and NELF-E; exosome-attracting factors ZCCHC7, ZCCHC8, and ZFC3H1; ARS2, which attracts processing and transport factors; and transport factor PHAX during the biogenesis of hTR. The data obtained revealed that ZFC3H1 participates in hTR biogenesis via pathways related to the polyadenylated RNA degradation mechanism. The data revealed essential differences that are important for understanding hTR biogenesis and that are interesting for further investigations of new, therapeutically significant targets.

Localization of mRNA is important in a number of cellular processes such as embryogenesis, cellular motility, polarity, and a variety of neurological processes. A synthetic device that controls cellular mRNA localization would facilitate investigations on the significance of mRNA localization in cellular function and allow an additional level of controlling gene expression. In this work, we developed the PUF (Pumilio and FBF homology domain)-assisted localization of RNA (PULR) system, which utilizes a eukaryotic cell's cytoskeletal transport machinery to reposition mRNA within a cell. Depending on the cellular motor used, we show ligand-dependent transport of mRNA toward either pole of the microtubular network of cultured cells. In addition, implementation of the reprogrammable PUF domain allowed the transport of untagged endogenous mRNA in primary neurons.

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.

The etiology of the autoimmune disorder systemic lupus erythematosus (SLE) remains poorly understood. In neuropsychiatric SLE (NPSLE), autoimmune responses against neural self-antigens find expression in neurological and cognitive alterations. SLE autoantibodies often target nucleic acids, including RNAs and specifically RNA domains with higher-order structural content. We report that autoantibodies directed against neuronal regulatory brain cytoplasmic (BC) RNAs were generated in a subset of SLE patients. By contrast, anti-BC RNA autoantibodies (anti-BC abs) were not detected in sera from patients with autoimmune diseases other than SLE (e.g., rheumatoid arthritis or multiple sclerosis) or in sera from healthy subjects with no evidence of disease. SLE anti-BC abs belong to the IgG class of immunoglobulins and target both primate BC200 RNA and rodent BC1 RNA. They are specifically directed at architectural motifs in BC RNA 5' stem-loop domains that serve as dendritic targeting elements (DTEs). SLE anti-BC abs effectively compete with RNA transport factor heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) for DTE access and significantly diminish BC RNA delivery to synapto-dendritic sites of function. In vivo experiments with male BALB/c mice indicate that, upon lipopolysaccharide-induced opening of the blood-brain barrier, SLE anti-BC abs are taken up by CNS neurons where they significantly impede localization of endogenous BC1 RNA to synapto-dendritic domains. Lack of BC1 RNA causes phenotypic abnormalities including epileptogenic responses and cognitive dysfunction. The combined data indicate a role for anti-BC RNA autoimmunity in SLE and its neuropsychiatric manifestations.SIGNIFICANCE STATEMENT Although clinical manifestations of neuropsychiatric lupus are well recognized, the underlying molecular-cellular alterations have been difficult to determine. We report that sera of a subset of lupus patients contain autoantibodies directed at regulatory brain cytoplasmic (BC) RNAs. These antibodies, which we call anti-BC abs, target the BC RNA 5' domain noncanonical motif structures that specify dendritic delivery. Lupus anti-BC abs effectively compete with RNA transport factor heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) for access to BC RNAs. As a result, hnRNP A2 is displaced, and BC RNAs are impaired in their ability to reach synapto-dendritic sites of function. The results reveal an unexpected link between BC RNA autoantibody recognition and dendritic RNA targeting. Cellular RNA dysregulation may thus be a contributing factor in the pathogenesis of neuropsychiatric lupus.

The precursors of RNP29 and Ferredoxin (Fd2) were previously identified in the cytosol of ppi2 plant cells with their N-terminal amino acid acetylated. Here, we explore whether precursor accumulation in ppi2 is characteristic for Toc159 client proteins, by characterizing the import properties of the RNP29 precursor in comparison to Fd2 and other Toc159-dependent or independent substrates. We find specific accumulation of the RNP29 precursor in ppi2 but not in wild type or ppi1 protoplasts. With the exception of Lhcb4, precursor accumulation is also detected with all other tested constructs in ppi2. However, RNP29 is clearly different from the other proteins because only precursor but almost no mature protein is detectable in protoplast extracts. Co-transformation of RNP29 with Toc159 complements its plastid import, supporting the hypothesis that RNP29 is a Toc159-dependent substrate. Exchange of the second amino acid in the RNP29 transit peptide to Glu or Asn prevents methionine excision but not N-terminal acetylation, suggesting that different N-acetyltransferases may act on chloroplast precursor proteins in vivo. All different RNP29 constructs are efficiently imported into wild type but not into ppi2 plastids, arguing for a minor impact of the N-terminal amino acid on the import process.

Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. Here, we show that in Drosophila the same machinery and RNA signals drive specific accumulation of maternal RNAs in the early oocyte and apical transcript localization in blastoderm embryos. We demonstrate in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos, and that interfering with the activities of Egl and BicD blocks apical localization. We propose that Egl and BicD are core components of a selective dynein motor complex that drives transcript localization in a variety of tissues.

Fission yeast Mei2p is an RNA-binding protein required for both premeiotic DNA synthesis and meiosis I. Mei2p binds to a polyadenylated RNA molecule, meiRNA, loss of which blocks meiosis I. Mei2p forms a dot in meiotic prophase nuclei. Here, we show that meiRNA is required for the nuclear localization of Mei2p and is detectable in the dot. However, Mei2p carrying a nuclear localization signal can produce a nuclear dot and promote meiosis I in the absence of meiRNA. Mei2p expressed in cultured mammalian cells stays in the cytoplasm, but it accumulates in the nucleolus if meiRNA is coexpressed. These results indicate that meiRNA contributes to the promotion of meiosis I exclusively as a cofactor that assists nuclear transport of Mei2p.

Most eukaryotic mRNAs are polyadenylated in the nucleus, and the poly(A)-tail is required for efficient mRNA export and translation. However, mechanisms governing mRNA transport remain unclear. Here, we report that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 acts as an energy sensor and negatively regulates poly(A)RNA transport via deacetylating a poly(A)-binding protein, PABP1. Upon energy starvation, SIRT1 interacts with and deacetylates PABP1 and deactivates its poly(A)RNA binding, leading to nuclear accumulation of PABP1 and poly(A)RNA and thus facilitating eukaryotic cells to attenuate protein synthesis and energy consumption to adapt to energy stress. Moreover, AMPK-directed SIRT1 phosphorylation is required for energy starvation-induced PABP1-SIRT1 association, PABP1 deacetylation, and poly(A)RNA nuclear retention. In addition, the SIRT1-PABP1 association is not specific to energy starvation but represents a common stress response. These observations provide insights into dynamic modulation of eukaryotic mRNA transport and translation, suggesting that the poly(A)-tail also provides a basis for eukaryotes to effectively shut down mature mRNA transport and thereby tailor protein synthesis to maintain energy homeostasis under stress conditions.

We previously identified an RNA transport element (RTE) present at a high copy number in the mouse genome. Here, we show that a related element, RTE-D, is part of a mobile LTR-retrotransposon, which belongs to a family of intracisternal A-particle related elements (IAP). We demonstrate that RTE-D is essential for the mobility of the retrotransposon and it can be substituted by other known RNA export signals. RTE-deficient IAP transcripts are retained in the nucleus, while the RTE-containing transcripts accumulate in the cytoplasm allowing Gag protein expression. RTE-D acts as a posttranscriptional control element in a heterologous reporter mRNA and is activated by the cellular RNA binding protein 15 (RBM15), as reported for the previously described RTE. We identified a complex family of RTE-containing IAPs in mouse and mapped the active RTE-D-containing IAPs to the Mmr10 group of LTR-retrotransposons. These data reveal that, despite a complex evolutionary history, retroelements and retroviruses share the dependency on posttranscriptional regulation.

Aichi virus (AiV), a small non-enveloped RNA virus, hijacks the endoplasmic reticulum (ER)-Golgi cholesterol transport machinery to form cholesterol-rich replication sites originating from Golgi membranes. Interferon-induced transmembrane proteins (IFITMs) are antiviral restriction factors, whose involvement in intracellular cholesterol transport is suggested. Here, we describe the roles of IFITM1 in cholesterol transport that affect AiV RNA replication. IFITM1 stimulated AiV RNA replication and its knockdown significantly reduced the replication. In replicon RNA-transfected or infected cells, endogenous IFITM1 localized to the viral RNA replication sites. Further, IFITM1 interacted with viral proteins and host Golgi proteins, ACBD3, PI4KB, OSBP, which constitute the replication sites. When overexpressed, IFITM1 localized to the Golgi as well as endosomes, and this phenotype was also observed for endogenous IFITM1 early in AiV RNA replication, leading to the distribution of cholesterol at the Golgi-derived replication sites. The pharmacological inhibition of ER-Golgi cholesterol transport or endosomal cholesterol export impaired AiV RNA replication and cholesterol accumulation at the replication sites. Such defects were corrected by expression of IFITM1. Overexpressed IFITM1 facilitated late endosome-Golgi cholesterol transport without any viral proteins. In summary, we propose a model in which IFITM1 enhances cholesterol transport to the Golgi to accumulate cholesterol at Golgi-derived replication sites, providing a novel mechanism by which IFITM1 enables efficient genome replication of non-enveloped RNA virus.

Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here, we show that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). We find that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. We additionally show that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. Our observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.

Pancreatic cancers are aggressive because they are highly invasive and highly metastatic; moreover, effective treatments for aggressive pancreatic cancers are lacking. Here, we report that the motor kinesin protein KIF20A promoted the motility and invasiveness of pancreatic cancer cells through transporting the RNA-binding protein IGF2BP3 and IGF2BP3-bound transcripts toward cell protrusions along microtubules. We previously reported that IGF2BP3 and its target transcripts are assembled into cytoplasmic stress granules of pancreatic cancer cells, and that IGF2BP3 promotes the motility and invasiveness of pancreatic cancer cells through regulation of localized translation of IGF2BP3-bound transcripts in cell protrusions. We show that knockdown of KIF20A inhibited accumulation of IGF2BP3-containing stress granules in cell protrusions and suppressed local protein expression from specific IGF2BP3-bound transcripts, ARF6 and ARHGEF4, in the protrusions. Our results provide insight into the link between regulation of KIF20A-mediated trafficking of IGF2BP3-containing stress granules and modulation of the motility and invasiveness in pancreatic cancers.

The development of the cerebral cortex requires balanced expansion and differentiation of neural stem/progenitor cells (NPCs), which rely on precise regulation of gene expression. Because NPCs often exhibit transcriptional priming of cell-fate-determination genes, the ultimate output of these genes for fate decisions must be carefully controlled in a timely fashion at the post-transcriptional level, but how that is achieved is poorly understood. Here, we report that de novo missense variants in an RNA-binding protein CELF2 cause human cortical malformations and perturb NPC fate decisions in mice by disrupting CELF2 nucleocytoplasmic transport. In self-renewing NPCs, CELF2 resides in the cytoplasm, where it represses mRNAs encoding cell fate regulators and neurodevelopmental disorder-related factors. The translocation of CELF2 into the nucleus releases mRNA for translation and thereby triggers NPC differentiation. Our results reveal that CELF2 translocation between subcellular compartments orchestrates mRNA at the translational level to instruct cell fates in cortical development.

Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species.

Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study.

In several Drosophila cell types, mRNA transport depends on microtubules, the molecular motor dynein and trans-acting factors including Egalitarian and Bicaudal-D. However, the molecular basis of transcript recognition by the localization machinery is poorly understood. Here, we characterize the features of hairy pair-rule RNA transcripts that mediate their apical localization, using in vivo injection of fluorescently labelled mRNAs into syncytial blastoderm embryos. We show that a 121-nucleotide element within the 3'-untranslated region is necessary and sufficient to mediate apical transport. The signal comprises two essential stem-loop structures, in which double-stranded stems are crucial for localization. Base-pair identities within the stems are not essential, but can contribute to the efficiency of localization, suggesting that specificity is mediated by higher-order structure. Using time-lapse microscopy, we measure the kinetics of localization and show that impaired localization of mutant signals is due to delayed formation of active motor complexes and, unexpectedly, to slower movement. These findings, and those from co-injecting wild-type and mutant RNAs, suggest that the efficiency of molecular motors is modulated by the character of their cargoes.

Sugar allocation between vegetative and reproductive tissues is vital to plant development, and sugar transporters play fundamental roles in this process. Although several transcription factors have been identified that control their transcription levels, the way in which the expression of sugar transporter genes is controlled at the posttranscriptional level is unknown. In this study, we showed that OsRRM, an RNA-binding protein, modulates sugar allocation in tissues on the source-to-sink route. The OsRRM expression pattern partly resembles that of several sugar transporter and transcription factor genes that specifically affect sugar transporter gene expression. The messenger RNA levels of almost all of the sugar transporter genes are severely reduced in the osrrm mutant, and this alters sugar metabolism and sugar signaling, which further affects plant height, flowering time, seed size, and starch synthesis. We further showed that OsRRM binds directly to messenger RNAs encoded by sugar transporter genes and thus may stabilize their transcripts. Therefore, we have uncovered the physiological function of OsRRM, which sheds new light on sugar metabolism and sugar signaling.

A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell's activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca(2+) levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca(2+) through voltage-dependent calcium channels upon β-adrenergic receptor activation. The combined data establish a functional correspondence between Ca(2+)-dependent RNA-protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.