Cleavage of Stau2 by 3C protease promotes EV-A71 replication.

BACKGROUND: Enterovirus A71 (EV-A71), as a neurotropic virus, mainly affects infants and young children under the age of 5. EV-A71 infection causes hand-foot-mouth disease and herpetic angina, and even life-threatening neurological complications. However, the molecular mechanism by which EV-A71 induces nervous system damage remains elusive. The viral protease 3C plays an important role during EV-A71 infection and is also a key intersection of virus-host interactions. Previously, we used yeast two-hybrid to screen out the host protein Double-stranded RNA-binding protein Staufen homolog 2 (Stau2), an important member involved in neuronal mRNA transport, potentially interacts with 3C. METHODS: We used coimmunoprecipitation (Co-IP) and immunofluorescence assay (IFA) to confirm that EV-A71 3C interacts with Stau2. By constructing the mutant of Stau2, we found the specific site where the 3C protease cleaves Stau2. Detection of VP1 protein using Western blotting characterized EV-A71 viral replication, and overexpression or knockdown of Stau2 exhibited effects on EV-A71 replication. The effect of different cleavage products on EV-A71 replication was demonstrated by constructing Stau2 truncates. RESULTS: In this study, we found that EV-A71 3C interacts with Stau2. Stau2 is cleaved by 3C at the Q507-G508 site. Overexpression of Stau2 promotes EV-A71 VP1 protein expression, whereas depletion of Stau2 by small interfering RNA inhibits EV-A71 replication. Stau2 is essential for EV-A71 replication, and the product of Stau2 cleavage by 3C, 508-570 aa, has activity that promotes EV-A71 replication. In addition, we found that mouse Stau2 is also cleaved by EV-A71 3C at the same site. CONCLUSIONS: Our research provides an example for EV-A71-host interaction, enriching key targets of host factors that contribute to viral replication.

RNF216 affects the stability of STAU2 in the hypothalamus.

Idiopathic hypogonadotropic hypogonadism (IHH) is a rare disease characterized by gonadal failure due to deficiency in gonadotropin-releasing hormone (GnRH) synthesis, secretion, or action. RNF216 variants have been recently identified in patients with IHH. Ring finger protein 216 (RNF216), as a ubiquitin E3 ligase, catalyzes the ubiquitination of target proteins with high specificity, which consequently modulates the stability, localization, and interaction of the target protein. In this study, we found that RNF216 interacted with Staufen2 (STAU2) and affected the stability of STAU2 through the ubiquitin-proteasome pathway. STAU2, as a double-stranded RNA-binding protein enriched in the nervous system, plays a role in RNA transport, RNA stability, translation, anchoring, and synaptic plasticity. Further, we revealed that STAU2 levels in the hypothalamus of RNF216-/- mice were increased compared with wild-type (WT) mice. The change in STAU2 protein homeostasis may affect a series of RNA cargoes. Therefore, we analyzed the changes in RNA levels in the hypothalamus of RNF216-/- mice and WT mice by RNA sequencing. We found that deletion of RNF216 led to decreased activities of the prolactin signaling pathway, neuroactive ligand-receptor interaction, GnRH signaling pathway, and ovarian steroidogenesis. The weakening of these signal pathways is likely to affect the secretion of GnRH, thereby affecting the development of gonads. Therefore, our study suggests that STAU2 may be a potential therapeutic target for IHH. Further experiments are needed to demonstrate the association between the weakening of these signaling pathways and the RNA-binding protein STAU2.

Systematic Identification of the RNA-Binding Protein STAU2 as a Key Regulator of Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a highly aggressive cancer. RNA-binding proteins (RBPs) regulate highly dynamic post-transcriptional processes and perform very important biological functions. Although over 1900 RBPs have been identified, most are considered markers of tumor progression, and further information on their general role in PAAD is not known. Here, we report a bioinformatics analysis that identified five hub RBPs and produced a high-value prognostic model based on The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets. Among these, the prognostic signature of the double-stranded RNA binding protein Staufen double-stranded RNA (STAU2) was identified. Firstly, we found that it is a highly expressed critical regulator of PAAD associated with poor clinical outcomes. Accordingly, the knockdown of STAU2 led to a profound decrease in PAAD cell growth, migration, and invasion and induced apoptosis of PAAD cells. Furthermore, through multiple omics analyses, we identified the key target genes of STAU2: Palladin cytoskeletal associated protein (PALLD), Heterogeneous nuclear ribonucleoprotein U (HNRNPU), SERPINE1 mRNA Binding Protein 1 (SERBP1), and DEAD-box polypeptide 3, X-Linked (DDX3X). Finally, we found that a high expression level of STAU2 not only helps PAAD evade the immune response but is also related to chemotherapy drug sensitivity, which implies that STAU2 could serve as a potential target for combinatorial therapy. These findings uncovered a novel role for STAU2 in PAAD aggression and resistance, suggesting that it probably represents a novel therapeutic and drug development target.

Staufen 1 is expressed by neural precursor cells in the developing murine cortex but is dispensable for NPC self-renewal and neuronal differentiation in vitro.

BACKGROUND: Proper development of the cerebral cortex relies on asymmetric divisions of neural precursor cells (NPCs) to produce a recurring NPC and a differentiated neuron. Asymmetric divisions are promoted by the differential localization of cell-fate determinants, such as mRNA, between daughter cells. Staufen 1 (Stau1) is an RNA-binding protein known to localize mRNA in mature hippocampal neurons. Its expression pattern and role in the developing mammalian cortex remains unknown. RESULTS: Both stau1 mRNA and Stau1 protein were found to be expressed in all cells of the developing murine cortex. Stau1 protein expression was characterized spatially and temporally throughout cortical development and found to be present in all stages investigated. We observed expression in the nucleus, cytoplasm and distal processes of both NPCs and newly born neurons and found it to shuttle between the nucleus and the cytoplasm. Upon shRNA-mediated knock-down of Stau1 in primary cultures of the developing cortex, we did not observe any phenotype in NPCs. They were able to both self-renew and generate neurons in the absence of Stau1 expression. CONCLUSIONS: We propose that Stau1 is either dispensable for the development of the cerebral cortex or that its paralogue, Stau2, is able to compensate for its loss.

STAU2 binds a complex RNA cargo that changes temporally with production of diverse intermediate progenitor cells during mouse corticogenesis.

STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.

Viral-Host Interactome Analysis Reveals Chicken STAU2 Interacts With Non-structural Protein 1 and Promotes the Replication of H5N1 Avian Influenza Virus.

As a highly pathogenic influenza virus, H5N1 avian influenza virus (AIV) poses a great threat to poultry production and public health. H5N1 AIV has a small genome and, therefore, relies heavily on its host cellular machinery to replicate. To develop a comprehensive understanding of how H5N1 AIV rewires host cellular machinery during the course of infection, it is crucial to identify which host proteins and complexes come into physical contact with the viral proteins. Here, we utilized affinity purification mass spectrometry (AP-MS) to systematically determine the physical interactions of 11 H5N1 AIV proteins with host proteins in chicken DF1 cells. We identified with high confidence 1,043 H5N1 AIV-chicken interactions involving 621 individual chicken proteins and uncovered a number of host proteins and complexes that were targeted by the viral proteins. Specifically, we revealed that chicken Staufen double-stranded RNA-binding protein 2 interacts with AIV non-structural protein 1 (NS1) and promotes the replication of the virus by enhancing the nuclear export of NS1 mRNA. This dataset facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of H5N1 AIV infection.

A retained intron in the 3'-UTR of Calm3 mRNA mediates its Staufen2- and activity-dependent localization to neuronal dendrites.

Dendritic localization and hence local mRNA translation contributes to synaptic plasticity in neurons. Staufen2 (Stau2) is a well-known neuronal double-stranded RNA-binding protein (dsRBP) that has been implicated in dendritic mRNA localization. The specificity of Stau2 binding to its target mRNAs remains elusive. Using individual-nucleotide resolution CLIP (iCLIP), we identified significantly enriched Stau2 binding to the 3'-UTRs of 356 transcripts. In 28 (7.9%) of those, binding occurred to a retained intron in their 3'-UTR The strongest bound 3'-UTR intron was present in the longest isoform of Calmodulin 3 (Calm3L ) mRNA Calm3L 3'-UTR contains six Stau2 crosslink clusters, four of which are in this retained 3'-UTR intron. The Calm3L mRNA localized to neuronal dendrites, while lack of the 3'-UTR intron impaired its dendritic localization. Importantly, Stau2 mediates this dendritic localization via the 3'-UTR intron, without affecting its stability. Also, NMDA-mediated synaptic activity specifically promoted the dendritic mRNA localization of the Calm3L isoform, while inhibition of synaptic activity reduced it substantially. Together, our results identify the retained intron as a critical element in recruiting Stau2, which then allows for the localization of Calm3L mRNA to distal dendrites.

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA.

Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.