N4BP1 mediates RAM domain-dependent notch signaling turnover during neocortical development.

Notch signaling pathway activity, particularly fluctuations in the biologically active effector fragment NICD, is required for rapid and efficient dynamic regulation of proper fate decisions in stem cells. In this study, we identified NEDD4-binding protein 1 (N4BP1), which is highly expressed in the developing mouse cerebral cortex, as a negative modulator of Notch signaling dynamics in neural progenitor cells. Intriguingly, N4BP1 regulated NICD stability specifically after Notch1 S3 cleavage through ubiquitin-mediated degradation that depended on its RAM domain, not its PEST domain, as had been extensively and previously described. The CoCUN domain in N4BP1, particularly the "Phe-Pro" motif (862/863 amino acid), was indispensable for mediating NICD degradation. The Ring family E3 ligase Trim21 was, in contrast to other NEDD4 family members, required for N4BP1-regulated NICD degradation. Overexpression of N4BP1 in cortical neural progenitors promoted neural stem cell differentiation, whereas neural progenitor cells lacking N4BP1 were sensitized to Notch signaling, resulting in the maintenance of stem-like properties in neural progenitor cells and lower production of cortical neurons.

Indispensable role of Nectin-like 4 in regulating synapse-related molecules, synaptic structure, and individual behavior.

Nectin-like family members (Necls) are involved in synaptic organization. In contrast to that of Necl-2/CADM1/SynCAM1, which is critical in synaptic events, investigation of Necl-4/CADM4/SynCAM4 in synapses has largely lagged behind given the particularity of homophilic self-interactions compared to interactions with other Necls. We sought to further understand the role of Necl-4 in synapses and found that knockout of Necl-4 led to aberrant expression levels of proteins mediating synaptic function in cortex homogenates and augmented accumulation of ionotropic glutamate receptor in postsynaptic density fractions, although a compensatory effect of Necl-1 on the expression levels existed. Concurrently, we also found increased synaptic clefts in the cortex and simplified dendritic morphology of primary cultured cortical neurons. Experiments on individual behaviors suggested that compared to their wild-type littermates, Necl-4-KO mice exhibited impaired acquisition of spatial memory and working memory and enhanced behavioral despair and anxiety-like behavior. These findings suggest that Necl-4 mediates synaptic function and related behaviors through an indispensable role and offer a new perspective about collaboration and specialization among Necls.

Comparison of the Spatiotemporal Expression Patterns of Three Cre Lines, Emx1IRES-Cre, D6-Cre and hGFAP-Cre, Commonly Used in Neocortical Development Research.

Emx1IRES-Cre, D6-Cre and hGFAP-Cre are commonly used to conditionally manipulate gene expression or lineage tracing because of their specificity in the dorsal telencephalon during early neurogenesis as previously described. However, the spatiotemporal differences in Cre recombinase activity would lead to divergent phenotypes. Here, we compared the patterns of Cre activity in the early embryos among the three lines by mating with reporter mice. The activities of Emx1IRES-Cre, D6-Cre and hGFAP-Cre were observed in the dorsal telencephalon, starting from approximately embryonic day 9.5, 11.5 and 12.5, respectively. Although all the three lines have activity in radial glial cells, Emx1IRES-Cre fully covers the dorsal and medial telencephalon, including the archicortex and cortical hem. D6-Cre is highly restricted to the dorsal telencephalon with anterior-low to posterior-high gradients, partially covers the hippocampus, and absent in the cortical hem. Moreover, both Emx1IRES-Cre and hGFAP-Cre exhibit Cre activity outside the dorsal neocortex. Meanwhile, we used the three Cre lines to mediate Dicer knockout and observed inconsistent phenotypes, including discrepancies in radial glial cell number, survival and neurogenesis in the neocortex and hippocampus. Together we proved differences in Cre activity can perturb the resultant phenotypes, which aid researchers in appropriate experimental design.

Zbed3 Is Indispensable for Wnt Signaling Regulation of Cortical Layers Formation in Developing Brain.

Wnt/ß-catenin signaling plays multiple important roles during mammalian brain development, and it regulates the proliferation and differentiation of neural progenitors in a context-dependent manner and affects neocortex layer formation. However, the specific role of Wnt/ß-catenin in neuronal layer fate determination in the neocortex is still unclear. Here, we report that Zbed3, which is a positive regulator of Wnt/ß-catenin signaling, colocalizes with ß-catenin at the endfeet of radial glia in the ventricular zone of embryo mouse neocortex. Overexpression and knockdown of Zbed3 increased and decreased the activity of Wnt/ß-catenin signaling in the neocortex, respectively. Interestingly, knockdown of Zbed3 in vivo could significantly shift neuronal fates from deep layers to upper layers but is not required for the proliferation and differentiation of neural progenitors. Overexpression of Zbed3 led to increased generation of deep-layer neurons without impairing cell cycle exit of neural progenitors. More importantly, knockdown of Zbed3 could effectively block the effects of the ectopic expression of stabilized ß-catenin on neocortex layer formation. Hence, our results demonstrate that Zbed3 is indispensable for Wnt/ß-catenin signaling regulating neuronal layer fates in the developing brain.

Structure of the heterophilic interaction between the nectin-like 4 and nectin-like 1 molecules.

Nectin-like (Necl) molecules are Ca2+-independent Ig-like transmembrane cell adhesion molecules that participate in junctions between different cell types. The specific cell-cell adhesions mediated by Necl proteins are important in neural development and have been implicated in neurodegenerative diseases. Here, we present the crystal structure of the mouse Necl-4 full ectodomain and the structure of the heterophilic Necl ectodomain complex formed by the mNecl-4 and mNecl-1 ectodomains. We demonstrate that, while the ectodomain of mNecl-4 is monomeric, it forms a stable heterodimer with Ig1 of mNecl-1, with an affinity significantly higher than that observed for self-dimerization of the mNecl-1 ectodomain. We validated our structural characterizations by performing a surface plasmon resonance assay and an Fc fusion protein binding assay in mouse primary dorsal root ganglia neurites and Schwann cells and identified a selection of residues important for heterophilic interactions. Finally, we proposed a model of Necl binding specificity that involves an induced-fit conformational change at the dimerization interface.

The spatiotemporal expression pattern of microRNAs in the developing mouse nervous system.

MicroRNAs (miRNAs) control various biological processes by inducing translational repression and transcript degradation of the target genes. In mammalian development, knowledge of the timing and expression pattern of each miRNA is important to determine and predict its function in vivo So far, no systematic analyses of the spatiotemporal expression pattern of miRNAs during mammalian neurodevelopment have been performed. Here, we isolated total RNAs from the embryonic dorsal forebrain of mice at different developmental stages and subjected these RNAs to microarray analyses. We selected 279 miRNAs that exhibited high signal intensities or ascending or descending expression dynamics. To ascertain the expression patterns of these miRNAs, we used locked nucleic acid (LNA)-modified miRNA probes in in situ hybridization experiments. Multiple miRNAs exhibited spatially restricted/enriched expression in anatomically distinct regions or in specific neuron subtypes in the embryonic brain and spinal cord, such as in the ventricular area, the striatum (and other basal ganglia), hypothalamus, choroid plexus, and the peripheral nervous system. These findings provide new insights into the expression and function of miRNAs during the development of the nervous system and could be used as a resource to facilitate studies in neurodevelopment.

Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C.

Extracellular vesicles (EV), as the intercellular information transfer molecules which can regulate the tumor microenvironment, promote migration and tumor progression. Previous studies reported that EV from endothelial cells was used to guide the fate and survival of gliomas, but many researches focus on normal human endothelial cells (NhEC) rather than tumor-derived endothelial cells. Our laboratory isolated human endothelial cells from glioma issue (GhEC). We have previously demonstrated that EV from GhEC and NhEC, which both can promote glioma stem cells (GSC) proliferation and tumorsphere formation in vitro and tumourigenicity in vivo by the transfer of CD9. However, NhEC-EV or GhEC-EV could suppress glioma cells (GC) proliferation in vitro. It demonstrates the undifferentiated impact of EV. Here, we first compared GhEC-EV proteins with NhEC-EV (Screening criteria: GhEC-EV/NhEC-EV, FC > 1.5), and obtained 70 differential expression proteins, most of which were associated with invasion and migration. We found that GhEC or GhEC-EV preferred promoting GC migration than treating with NhEC or NhEC-EV. In terms of mechanism, we further revealed that EV-mediated transfer of MYO1C induced glioma cell LN229 migration. Knockdown of MYO1C in GhEC or GhEC-EV suppressed this effect. Overexpression of MYO1C promoted migration on the contrary. MYO1C was also detected in glioma cerebrospinal fluid (CSF), which is more suitable as a liquid biopsy biomarker and contributes to early diagnosis and monitoring in glioma. Our findings provide a new protein-MYO1C in EV to target tumor blood vessels, and bring a new point-cut to the treatment of gliomablastoma (GBM).

PTB-AS, a Novel Natural Antisense Transcript, Promotes Glioma Progression by Improving PTBP1 mRNA Stability with SND1.

Glioma, the most common primary malignancy in the brain, has high recurrence and lethality rates, and thus, elucidation of the molecular mechanisms of this incurable disease is urgently needed. Poly-pyrimidine tract binding protein (PTBP1, also known as hnRNP I), an RNA-binding protein, has various mechanisms to promote gliomagenesis. However, the mechanisms regulating PTBP1 expression are unclear. Herein, we report a novel natural antisense noncoding RNA, PTB-AS, whose expression correlated positively with PTBP1 mRNA. We found that PTB-AS significantly promoted the proliferation and migration in vivo and in vitro of glioma cells. PTB-AS substantially increased the PTBP1 level by directly binding to its 3' UTR and stabilizing the mRNA. Furthermore, staphylococcal nuclease domain-containing 1 (SND1) dramatically increased the binding capacity between PTB-AS and PTBP1 mRNA. Mechanistically, PTB-AS could mask the binding site of miR-9 in the PTBP1-3' UTR; miR-9 negatively regulates PTBP1. To summarize, we revealed that PTB-AS, which maintains the PTBP1 level through extended base pairing to the PTBP1 3' UTR with the assistance of SND1, could significantly promote gliomagenesis.

Long noncoding RNA HOXD-AS2 regulates cell cycle to promote glioma progression.

Now, numerous exciting findings have been found that long noncoding RNAs (lncRNAs) play a vital role in cancer malignant progression. However, their potential involvement in glioma is not well understood. Here, we performed a high-throughput microarray to detect the lncRNA expression profiles between glioma cell lines and normal astrocyte cell lines. HOXD-AS2 was increased in glioma cells and it was associated with glioma grade and poor prognosis. Loss of HOXD-AS2 can inhibit glioma cell growth by inducing cell-cycle G1 arrest in vitro. The proliferation of glioma was inhibited followed by knocking down the expression of HOXD-AS2 not only in subcutaneous injection model but also in orthotopic implantation model. These findings indicate that HOXD-AS2 promotes the glioma progression and may serve as a potential target for glioma diagnosis and therapy.

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9.

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

PTBP1 induces ADAR1 p110 isoform expression through IRES-like dependent translation control and influences cell proliferation in gliomas.

Internal ribosomal entry site (IRES)-mediated translation initiation is constitutively activated during stress conditions such as tumorigenesis and hypoxia. The RNA editing enzyme ADAR1 plays an important role in physiology and pathology. Initially, we found that the ADAR1 p150 or p110 transcript levels were decreased in glioma cells compared with normal astrocyte cells. In contrast, protein levels of ADAR1 p110 were significantly upregulated in glioma tissues and cells. This expression pattern indicated translationally controlled regulation. We identified an 885-nt sequence that was located between AUG1 and AUG2 within the ADAR1 mRNA that exhibited IRES-like activity. Furthermore, we confirmed that the translational mode of ADAR1 p110 was mediated by PTBP1 in glioma cells. The protein levels of PTBP1 and ADAR1 were cooperatively expressed in glioma tissues and cells. Knocking down ADAR1 p110 significantly decreased cell proliferation in three types of glioma cells (T98G, U87MG and A172). The removal of a minimal IRES-like sequence in a p150-overexpression construct could effectively abolish p110 induction and resulted in the slight suppression of cell proliferation compared with ADAR1-p150 overexpression in siPTBP1-treated T98G cells. In summary, our study revealed a mechanism whereby ADAR1 p110 can be activated by PTBP1 through an IRES-like element in glioma cells, and ADAR1 is essential for the maintenance of gliomagenesis.

Proteomic screening and identification of microRNA-128 targets in glioma cells.

Brain-enriched miR-128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR-128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR-128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR-128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR-128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR-128 were screened and laid the foundation for research into the miR-128 regulation network.

cAMP response element-binding protein promotes gliomagenesis by modulating the expression of oncogenic microRNA-23a.

Gliomas are the most common and deadly type of primary brain tumor. In this study, we showed that cAMP response element-binding protein (CREB), a proto-oncogenic transcription factor that is overexpressed in gliomas, can promote gliomagenesis by modulating the expression of oncogenic microRNA-23a (mir-23a). First, we found that CREB is highly expressed in glioma tissues and cell lines. CREB is also essential for glioma cell growth and cell survival in vitro and is critical for gliomagenesis in vivo. Second, microRNA microarray, ChIP-chip, ChIP-quantitative PCR, and luciferase reporter assays showed that CREB directly binds to the regulatory sequences of mir-23a and enhance the expression of mir-23a. Moreover, mir-23a was confirmed as a functional downstream target of CREB in glioma cell growth and cell survival. Finally, using computational prediction followed by experimental confirmation, we identified PTEN, which is frequently silenced in gliomas, as a downstream target of mir-23a. Taken together, we propose that CREB promotes gliomagenesis and acts as a modulator of oncogenic mir-23a, which represses the tumor suppressor PTEN.

RAB31 in glioma-derived endothelial cells promotes glioma cell invasion via extracellular vesicle-mediated enrichment of MYO1C.

Extracellular vesicles (EV), important messengers in intercellular communication, can load and transport various bioactive components and participate in different biological processes. We previously isolated glioma human endothelial cells (GhECs) and found that GhECs, rather than normal human brain endothelial cells (NhECs), exhibit specific enrichment of MYO1C into EVs and promote the migration of glioma cells. In this study, we explored the mechanism by which MYO1C is secreted into EVs. We report that such secretion is dependent on RAB31, RAB27B, and FAS. When expression of RAB31 increases, MYO1C is enriched in secretory EVs. Finally, we identified an EV export mechanism for MYO1C that promotes glioma cell invasion and is dependent on RAB31 in GhECs. In summary, our data indicate that the knockdown of RAB31 can reduce enrichment of MYO1C in extracellular vesicles, thereby attenuating the promotion of glioma cell invasion by GhEC-EVs.

Temporal transcriptomic dynamics in developing macaque neocortex.

Despite intense research on mice, the transcriptional regulation of neocortical neurogenesis remains limited in humans and non-human primates. Cortical development in rhesus macaque is known to recapitulate multiple facets of cortical development in humans, including the complex composition of neural stem cells and the thicker supragranular layer. To characterize temporal shifts in transcriptomic programming responsible for differentiation from stem cells to neurons, we sampled parietal lobes of rhesus macaque at E40, E50, E70, E80, and E90, spanning the full period of prenatal neurogenesis. Single-cell RNA sequencing produced a transcriptomic atlas of developing parietal lobe in rhesus macaque neocortex. Identification of distinct cell types and neural stem cells emerging in different developmental stages revealed a terminally bifurcating trajectory from stem cells to neurons. Notably, deep-layer neurons appear in the early stages of neurogenesis, while upper-layer neurons appear later. While these different lineages show overlap in their differentiation program, cell fates are determined post-mitotically. Trajectories analysis from ventricular radial glia (vRGs) to outer radial glia (oRGs) revealed dynamic gene expression profiles and identified differential activation of BMP, FGF, and WNT signaling pathways between vRGs and oRGs. These results provide a comprehensive overview of the temporal patterns of gene expression leading to different fates of radial glial progenitors during neocortex layer formation.

Docking protein 6 (DOK6) selectively docks the neurotrophic signaling transduction to restrain peripheral neuropathy.

The appropriate and specific response of nerve cells to various external cues is essential for the establishment and maintenance of neural circuits, and this process requires the proper recruitment of adaptor molecules to selectively activate downstream pathways. Here, we identified that DOK6, a member of the Dok (downstream of tyrosine kinases) family, is required for the maintenance of peripheral axons, and that loss of Dok6 can cause typical peripheral neuropathy symptoms in mice, manifested as impaired sensory, abnormal posture, paw deformities, blocked nerve conduction, and dysmyelination. Furthermore, Dok6 is highly expressed in peripheral neurons but not in Schwann cells, and genetic deletion of Dok6 in peripheral neurons led to typical peripheral myelin outfolding, axon destruction, and hindered retrograde axonal transport. Specifically, DOK6 acts as an adaptor protein for selectivity-mediated neurotrophic signal transduction and retrograde transport for TrkC and Ret but not for TrkA and TrkB. DOK6 interacts with certain proteins in the trafficking machinery and controls their phosphorylation, including MAP1B, Tau and Dynein for axonal transport, and specifically activates the downstream ERK1/2 kinase pathway to maintain axonal survival and homeostasis. This finding provides new clues to potential insights into the pathogenesis and treatment of hereditary peripheral neuropathies and other degenerative diseases.

Targeting HOTAIRM1 ameliorates glioblastoma by disrupting mitochondrial oxidative phosphorylation and serine metabolism.

Serine hydroxymethyltransferase 2 (SHMT2), which catalyzes the conversion of serine to glycine and one-carbon transfer reactions in mitochondria, is significantly upregulated in glioblastoma (GBM). However, the mechanism by which the stability of SHMT2 gene expression is maintained to drive GBM tumorigenesis has not been clarified. Herein, through microarray screening, we identified that HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) modulates the SHMT2 level in various GBM cell lines. Serine catabolism and mitochondrial oxidative phosphorylation activities were decreased by HOTAIRM1 inhibition. Mechanistically, according to our mass spectrometry and eCLIP-seq results, HOTAIRM1 can bind to PTBP1 and IGF2BP2. Furthermore, HOTAIRM1 maintains the stability of SHMT2 by promoting the recognition of an m6A site and the interaction of PTBP1/IGF2BP2 with SHMT2 mRNA. The stability of HOTAIRM1 can also be enhanced and results in positive feedback regulation to support the progression of GBM. Thus, targeting HOTAIRM1 could be a promising metabolic therapy for GBM.

The Ash2l SDI Domain Is Required to Maintain the Stability and Binding of DPY30.

ASH2L and DPY30 are important for the assembly and catalytic activity of the complex associated with SET1 (COMPASS), which catalyzes histone methylation and regulates gene expression. However, the regulations among COMPASS components are not fully understood. Here, we leveraged a mouse model and cell lines to observe the outcome of Ash2l depletion and found a significant decrease in DPY30. Analyzing ASH2L ChIP-seq and RNA-seq data excluded transcriptional and translational regulation of ASH2L to DPY30. The decrease in DPY30 was further attributed to the degradation via the ubiquitin-mediated proteasomal pathway. We also verified that three amino acids in the ASH2L Sdc1 DPY30 interaction (SDI) domain are essential for the recognition and binding of DPY30. Lastly, we unexpectedly observed that overexpression of DPY30 in Ash2l-depleted cells rescued the decrease in Ccnd1 and the abnormal cell cycle, which indicates that DPY30 can participate in other complexes to regulate gene expression. Overall, our results, for the first time, reveal that the existence of DPY30 relies on the binding with ASH2L, with degradation of DPY30 via the ubiquitin-proteasome system, and they further indicate that the function of DPY30 can be independent of ASH2L.

Long Non-coding RNA T-uc.189 Modulates Neural Progenitor Cell Fate by Regulating Srsf3 During Mouse Cerebral Cortex Development.

Neurogenesis is a complex process that depends on the delicate regulation of spatial and temporal gene expression. In our previous study, we found that transcribed ultra-conserved regions (T-UCRs), a class of long non-coding RNAs that contain UCRs, are expressed in the developing nervous systems of mice, rhesus monkeys, and humans. In this study, we first detected the full-length sequence of T-uc.189, revealing that it was mainly concentrated in the ventricular zone (VZ) and that its expression decreased as the brain matured. Moreover, we demonstrated that knockdown of T-uc.189 inhibited neurogenesis. In addition, we found that T-uc.189 positively regulated the expression of serine-arginine-rich splicing factor 3 (Srsf3). Taken together, our results are the first to demonstrate that T-uc.189 regulates the expression of Srsf3 to maintain normal neurogenesis during cortical development.

A single mutation in the cis-acting replication element identified within the EV-A71 2C-coding region causes defects in virus production in cell culture.

ABSTRACTEnterovirus A71 (EV-A71) can cause hand, foot and mouth disease with neurological and systemic complications, most frequently affecting children and infants. We describe a cis-acting replication element (cre) with a conserved stem-loop structure within the EV-A71 2C-coding region. By site-directed mutagenesis and reverse genetics using the EV-A71 full-length genome and the EV-A71 replicon containing the firefly luciferase reporter gene in place of the P1 region, the stem-loop structure and the AAACA in the loop of the cre were confirmed to be required for the EV-A71 replication phenotype. EV-A71 genomes containing a mutation at the first or third A residue of AAACA could not be recovered. Insertion of a wild-type cre from EV-A71 or poliovirus in the 5'UTR led to successful recovery of the replication of nonviable mutants. Furthermore, the cre mutants showed lower binding capacity with the host cellular factor IGF2BP2, knockdown of which resulted in a significant decrease in EV-A71 production. All the available evidence shows the location independence but functional importance of the interaction of the cre with the cellular host for efficient production of EV-A71, contributing to the growing body of knowledge regarding picornavirus cres.