A conserved role for the zinc finger polyadenosine RNA binding protein,ZC3H14, in control of poly(A) tail length

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.     

The Drosophila ortholog of the Zc3h14 RNA binding protein acts within neurons to pattern axon projection in the developing brain

The dNab2 polyadenosine RNA binding protein is the D. melanogaster ortholog of the vertebrate ZC3H14 protein, which is lost in a form of inherited intellectual disability (ID). Human ZC3H14 can rescue D. melanogaster dNab2 mutant phenotypes when expressed in all neurons of the developing nervous system, suggesting that dNab2/ZC3H14 performs well‐conserved roles in neurons. However, the cellular and molecular requirements for dNab2/ZC3H14 in the developing nervous system have not been defined in any organism. Here we show that dNab2 is autonomously required within neurons to pattern axon projection from Kenyon neurons into the mushroom bodies, which are required for associative olfactory learning and memory in insects. Mushroom body axons lacking dNab2 project aberrantly across the brain midline and also show evidence of defective branching. Coupled with the prior finding that ZC3H14 is highly expressed in rodent hippocampal neurons, this requirement for dNab2 in mushroom body neurons suggests that dNab2/ZC3H14 has a conserved role in supporting axon projection and branching. Consistent with this idea, loss of dNab2 impairs short‐term memory in a courtship conditioning assay. Taken together these results reveal a cell‐autonomous requirement for the dNab2 RNA binding protein in mushroom body development and provide a window into potential neurodevelopmental functions of the human ZC3H14 protein. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 93–106, 2016     

Transportin 2 regulates apoptosis through the RNA-binding protein HuR

In response to severe stress, apoptotic cell death is engaged. Apoptosis is a well orchestrated process that involves the activation and implication of many factors. In this study, we identified a role for the nuclear trafficking factor TRN2 (transportin 2) in cell death. TRN2 is normally responsible for the nuclear import of the RNA-binding protein HuR. During apoptosis, however, HuR accumulates in the cytoplasm. This is due to the caspase-mediated cleavage of the cytoplasmic fraction of HuR. One of the cleavage fragments generated by this processing of HuR interacts with TRN2 and thus blocks the re-import of HuR into the nucleus. This concentrates HuR in the cytoplasm, advancing apoptosis. Therefore, increasing or decreasing the levels of TRN2 has an inverse consequential effect on cell death, demonstrating for the first time the role of a nucleocytoplasmic transport factor in apoptosis.     

IGF2BP2, an RNA-binding protein regulates cell proliferation and osteogenic differentiation by stabilizing SRF mRNA

Osteoblast proliferation and osteogenic differentiation (OGD) are regulated by complex mechanisms. The roles in cell proliferation and OGD of RNA-binding proteins in the insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family remain unclear. To elucidate this, we examined the differential expression of IGF2BP2 in OGD and osteoporosis, and the expression profile of IGF2BP2-binding RNA in vitro. We screened the GEO database for differential expression of IGF2BP in OGD and osteoporosis, and verified the RNAs interacting with IGF2BP2 via RNA immunoprecipitation sequencing assays. The proliferation and OGD of IGF2BP2- and serum response factor (SRF)-treated cells, and their regulatory mechanisms, were examined. IGF2BP2 was differentially expressed in OGD and osteoporosis. The RNA immunoprecipitation sequencing assay identified all of the RNAs that bind with IGF2BP2, and revealed SRF as a target of IGF2BP2. IGF2BP2 and SRF inhibition impaired MC3T3-E1 cell growth but promoted OGD. The mRNA stability analysis revealed that IGF2BP2 enhanced SRF mRNA stability against degradation. In summary, IGF2BP2 is a potential biomarker and therapeutic target for osteoporosis and OGD.     

Leafy head2, which encodes a putative RNA-binding protein, regulates shoot development of rice

During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 （lhd2） mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TEl. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOXand hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass.     

The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor

The cold-inducible RNA-binding protein (CIRP) is a nuclear 18-kDa protein consisting of an amino-terminal RNA Recognition Motif (RRM) and a carboxyl-terminal domain containing several RGG motifs. First characterized for its overexpression upon cold shock, CIRP is also induced by stresses such as UV irradiation and hypoxia. Here, we investigated the expression as well as the subcellular localization of CIRP in response to other stress conditions. We demonstrate that oxidative stress leads to the migration of CIRP to stress granules (SGs) without alteration of expression. Stress granules are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. Relocalization of CIRP into SGs also occurs upon other cytoplasmic stresses (osmotic pressure or heat shock) as well as in response to stresses of the endoplasmic reticulum. CIRP migration into SGs is independent from TIA-1 which has been previously reported to be a general mediator of SG formation, thereby suggesting the existence of multiple pathways leading to SG formation. Moreover, deletion mutants revealed that both RGG and RRM domains can independently promote CIRP migration into SGs. However, the methylation of arginine residues in the RGG domain is necessary for CIRP to exit the nucleus to be further recruited into SGs. By RNA-tethering experiments, we also show that CIRP down-regulates mRNA translation and that this activity is carried by the carboxyl-terminal RG-enriched domain. Altogether, our findings further reveal the diversity of mechanisms by which CIRP is regulated by environmental stresses and provide new insights into CIRP cytoplasmic function.     

Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome

Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age‐related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since carbonyl groups are not present in the 20 canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin beads. On‐bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ MS‐based proteomics approach. Western blotting and biotin quantitation assay approaches were also investigated. By both Western blotting and proteomics, Paraquat exposure, but not Sod2 knockdown, resulted in increased carbonylated protein relative abundance. For Paraquat exposure versus control, the median carbonylated protein relative abundance ratio (1.53) determined using MS‐based proteomics was in good agreement with that obtained using a commercial biotin quantitation kit (1.36).     

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.Supplementary key words: low density lipoprotein receptor, receptors, protein trafficking, cholesterol, lipoproteins, CRISPR screen, HuH7 cells, endocytosis, RAB10, RAB11

An elevated level of circulating low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic cardiovascular diseases, including myocardial infarction and stroke (1, 2, 3, 4, 5, 6, 7). Regulation of plasma cholesterol is governed by a complex interplay between dietary absorption, de novo biosynthesis, and clearance from the bloodstream. Therapeutic targeting of LDL clearance has been a highly successful strategy for the prevention and treatment of atherosclerosis. LDL clearance is mediated by the LDL receptor (LDLR), a cell-surface glycoprotein that directly binds to the apolipoprotein B component of LDL particles and triggers clathrin-mediated endocytosis. The acidic environment of the endosomal lumen induces complex dissociation, with LDL subsequently transported to the lysosome for hydrolysis, and free LDLR recycled back to the plasma membrane (8, 9). Many regulatory proteins affecting the endocytic pathway and cell-surface expression of LDLR have been identified, including PCSK9, a negative regulator that redirects LDLR to the lysosome for degradation (10), and IDOL, a ubiquitin ligase that induces proteasomal degradation of LDLR (11, 12). Although much is known about the regulation of LDLR expression and endocytosis, questions remain concerning the molecular determinants of LDLR recycling.We recently reported a genome-wide CRISPR screen for modifiers of LDL uptake in HuH7 cells (13). This screen identified RAB10, a small GTPase known to mediate trafficking of vesicles between intracellular compartments, as a key regulator of LDL uptake. Deletion of RAB10 decreased cellular endocytosis of LDL but increased accumulation of another endocytic cargo, transferrin. The receptors for LDL (LDLR) and transferrin receptor (TFR) are both endocytosed from the cell surface via clathrin-coated vesicles and transported through intracellular recycling pathways (14, 15, 16, 17, 18, 19, 20). In this study, we investigated the role of RAB10 in LDL and transferrin endocytosis. Our results demonstrate that GTP-bound RAB10 positively regulates the activity of LDLR and TFR by accelerating the recycling of both proteins to the plasma membrane.     

The dynamics of the proteome: strategies for measuring protein turnover on a proteome-wide scale.

Quantitative proteomics captures the steady-state amount of a protein in a cell but does not explain how a change in protein amount is manifest -- whether through a change in synthesis or a change in degradation. If we are to understand the changes in the proteome, we will need to define such processes. In this brief review, strategies for the determination of intracellular protein dynamics on a proteome-wide scale are discussed.     

Expression of RNA-binding protein Rbfox1l demarcates a restricted population of dorsal telencephalic neurons within the adult zebrafish brain

Rbfox RNA-binding proteins are expressed in the adult mammalian brain and are required for proper brain development and function. Studies in mice and humans have implicated Rbfox1/RBFOX1 in autism, neuronal excitation and epilepsy, and Rbfox2/RBFOX2 in cerebellar development. The zebrafish has emerged as a prominent model system for brain study, possessing neuroanatomical conservation with mammals and an extensive capacity for adult neurogenesis and plasticity. In this study, we characterize Rbfox1l and Rbfox2 expression in the adult zebrafish brain. While Rbfox2 is expressed broadly, Rbfox1l is expressed in restricted populations of neurons in the dorsal telencephalon and cerebellum. In the dorsal telencephalon, Rbfox1l is expressed in a specific population of neurons spanning Dm and Dc regions. In the cerebellum, Rbfox1l and Rbfox2 are expressed in the Purkinje cell layer, reminiscent of Rbfox1 and Rbfox2 expression in the mammalian cerebellum. Our findings motivate future studies of Rbfox function in the zebrafish brain.     

Structure of the N-terminal Mlp1-binding domain of the Saccharomyces cerevisiae mRNA-binding protein, Nab2

Nuclear abundant poly(A) RNA-binding protein 2 (Nab2) is an essential yeast heterogeneous nuclear ribonucleoprotein that modulates both mRNA nuclear export and poly(A) tail length. The N-terminal domain of Nab2 (residues 1-97) mediates interactions with both the C-terminal globular domain of the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), and the mRNA export factor, Gfd1. The solution and crystal structures of the Nab2 N-terminal domain show a primarily helical fold that is analogous to the PWI fold found in several other RNA-binding proteins. In contrast to other PWI-containing proteins, we find no evidence that the Nab2 N-terminal domain binds to nucleic acids. Instead, this domain appears to mediate protein:protein interactions that facilitate the nuclear export of mRNA. The Nab2 N-terminal domain has a distinctive hydrophobic patch centered on Phe73, consistent with this region of the surface being a protein:protein interaction site. Engineered mutations within this hydrophobic patch attenuate the interaction with the Mlp1 C-terminal domain but do not alter the interaction with Gfd1, indicating that this patch forms a crucial component of the interface between Nab2 and Mlp1.     

The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.     

The Arabidopsis ortholog of the 77 kDa subunit of the cleavage stimulatory factor (AtCstF-77) involved in mRNA polyadenylation is an RNA-binding protein

The 77 kDa subunit of the polyadenylation cleavage stimulation factor (CstF77) is important in messenger RNA 3′ end processing. Previously, we demonstrated that AtCstF77 interacts with AtCPSF30, the Arabidopsis ortholog of the 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. In further dissecting this interaction, it was found that the C-terminus of AtCstF77 interacts with AtCPSF30. Remarkably, we also found that the C-terminal domain of AtCstF77 possesses RNA-binding ability. These studies therefore reveal AtCstF77 to be an RNA-binding protein, adding yet another RNA-binding activity to the plant polyadenylation complex. This raises interesting questions as to the means by which RNAs are recognized during mRNA 3′ end formation in plants.

Structured summary:

MINT-7712550: AtCstF77 (uniprotkb:Q8LKG5) binds (MI:0407) to AtCPSF30 (uniprotkb:A9LNK9) by pull down (MI:0096)     

S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.     

The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.