poly(A)-binding Protein Research Articles

The bent conformation of poly(A)-binding protein induced by RNA-binding is required for its translational activation function

ABSTRACTA recent study revealed that poly(A)-binding protein (PABP) bound to poly(A) RNA exhibits a sharply bent configuration at the linker region between RNA-recognition motif 2 (RRM2) and RRM3, whereas free PABP exhibits a highly flexible linear configuration. However, the physiological role of the bent structure of mRNA-bound PABP remains unknown. We investigated a role of the bent structure of PABP by constructing a PABP variant that fails to form the poly(A)-dependent bent structure but maintains its poly(A)-binding activity. We found that the bent structure of PABP/poly(A) complex is required for PABP's efficient interaction with eIF4G and eIF4G/eIF4E complex. Moreover, the mutant PABP had compromised translation activation function and failed to augment the formation of 80S translation initiation complex in an in vitro translation system. These results suggest that the bent conformation of PABP, which is induced by the interaction with 3′ poly(A) tail, mediates poly(A)-dependent translation by facilitating the interaction with eIF4G and the eIF4G/eIF4E complex. The preferential binding of the eIF4G/eIF4E complex to the bent PABP/poly(A) complex seems to be a mechanism discriminating the mRNA-bound PABPs participating in translation from the idling mRNA-unbound PABPs.

Interacting protein partners of Arabidopsis RNA-binding protein AtRBP45b.

RNA binding proteins, important players in post-transcriptional gene regulation, usually exist in ribonuclear complexes. However, even in model systems like Arabidopsis characterisation of RBP associated proteins is limited. In this study, we investigated the interacting proteins of the Arabidopsis AtRBP45b, which is involved in stress signalling. In vivo localisation of AtRBP45b was conducted using 35S-GFP. FLAG-tagged AtRBP45b under control of the 35S promoter in the Atrbp45b-1 mutant background was used to pull down AtRBP45b interacting proteins. Yeast two-hybrid analysis, fluorescence energy resonance transfer assays were used to confirm the veracity of the AtRBP45b interacting proteins. In planta GFP-tagging indicated AtRBP45b is localised to the nucleus and the cytosol.AtRBP45b protein has a N-terminal proline-rich region and a C-terminal glutamine-rich domain that are usually involved in protein-protein interactions. Co-immunoprecipitation followed by mass spectrometry-based protein sequencing led to identification of 30 proteins that interacted with AtRBP45b. Using information from interactome databases (BIOGRID, INTACT and STRING), pull-down assays and localisation data, 12 putative interacting proteins were selected for yeast two-hybrid analysis. Cap-binding protein (CBP20, At5g44200) and polyA-binding protein (PAB8, At1g49760) were shown to interact with AtRBP45b. Based on its interacting partners we speculate that AtRBP45b may play an important role in RNA metabolism, especially in aspects related to mRNA stability and translation initiation during stress conditions in plants.

The Aspergillus nidulans Pbp1 homolog is required for normal sexual development and secondary metabolism

P bodies and stress granules are RNA-containing structures governing mRNA degradation and translational arrest, respectively. Saccharomyces cerevisiae Pbp1 protein localizes to stress granules and promotes their formation and is involved in proper polyadenylation, suppression of RNA-DNA hybrids, and preventing aberrant rDNA recombination. A genetic screen for Aspergillus nidulans mutants aberrant in secondary metabolism identified the Pbp1 homolog, PbpA. Using Dcp1 (mRNA decapping) as a marker for P-body formation and FabM (Pab1, poly-A binding protein) to track stress granule accumulation, we examine the dynamics of RNA granule formation in A. nidulans cells lacking pub1, edc3, and pbpA. Although PbpA acts as a functional homolog of yeast PBP1, PbpA had little impact on either P-body or stress granule formation in A. nidulans in contrast to Pub1 and Edc3. However, we find that PbpA is critical for sexual development and its loss increases the production of some secondary metabolites including the carcinogen sterigmatocystin.

Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development.

The poly(A)-binding protein (PABP) binds to the poly(A) tail of eukaryotic cellular mRNAs and contributes to their stability and translational efficiency. In plants, PABP is expressed from an unusually large gene family grouped into 3 classes that expanded during the evolution of land plants. Subsequent to expansion of the family, members diverged in their primary sequence and in expression. Further expansion of the family and divergence of its members in the Brassicaceae demonstrate the continued dynamic evolution of PABP in plants. In this study, the function of the widely-expressed class II PABP family members was examined to determine how individual class II members contribute to plant growth and development. Of the 3 class II PABP members, PAB2 and PAB4 contribute most to vegetative growth and vegetative-to-floral transition whereas PAB2, and the recently-evolved third class II member, PAB8, contribute to inflorescence and silique growth. Interestingly, although class I and class III PABP members are expressed specifically in reproductive organs, class II PABP members are also necessary for fertility in that the combinatorial loss of PAB2 and either PAB4 or PAB8 expression resulted in reduced fertility. Although all 3 class II members are required for protein expression, PAB4 contributes most to the steady-state level of a reporter mRNA and to protein expression. These findings suggest that class II PABP members are partially overlapping in function but also involved in distinct aspects of plant growth and development.

The role of the pAbp gene encoding the cytoplasmic poly(A)-binding protein in spermatogenesis of Drosophila melanogaster

The Drosophila melanogaster pAbp gene encodes the cytoplasmic poly(A)-binding protein (PABP). Cytoplasmic PABPs participate in the metabolic pathways of the mRNA: translation initiation and termination, cytoplasmic polyadenylation/deadenylation, mRNA stability, mRNA degradation. Despite the extensive biochemical and structural characterization, relatively little is known about the biological roles of PABPs in the processes of cellular development and differentiation. In Drosophila spermatogenesis, posttranscriptional mechanisms of gene regulation play an important role, so cytoplasmic PABP can have significant function in this process. Deletion of PABP leads to embryonic lethality. However, some flies carrying combinations of mutant pAbp alleles survive but display male sterility and show defects in spermatogenesis. It has previously been shown that hypomorphic pAbp mutations cause a number of meiotic defects, abnormalities of Nebenkern formation. These data provide an insight into the effect of pAbp mutation on the individual events of spermatogenesis, but they do not cover the entire process. We studied spermatogenesis in pAbp allele heterozygotes by transmission electron and fluorescent light microscopy. We showed that cellular mitochondria were the primary structural target of the mutation. Abnormal mitochondria were less structured, swollen, had transparent matrix and depleted cristae. Further mutation had a polymorphous effect and induced anomalies in the ultrastructure of mature spermatocytes, defects in Nebenkern formation and division, axial complex formation, shutdown of spermatogenesis during spermatid elongation. Thus our data show a significant role of PABP in structural transformations of male germ cells during entire spermato-genesis.

Poly(A)-binding proteins are required for translational regulation in vertebrate oocytes and early embryos.

Poly(A)-binding proteins (PABPs) function in the timely regulation of gene expression during oocyte maturation, fertilisation and early embryo development in vertebrates. To this end, PABPs bind to poly(A) tails or specific sequences of maternally stored mRNAs to protect them from degradation and to promote their translational activities. To date, two structurally different PABP groups have been identified: (1) cytoplasmic PABPs, including poly(A)-binding protein, cytoplasmic 1 (PABPC1), embryonic poly(A)-binding protein (EPAB), induced PABP and poly(A)-binding protein, cytoplasmic 3; and (2) nuclear PABPs, namely embryonic poly(A)-binding protein 2 and nuclear poly(A)-binding protein 1. Many studies have been undertaken to characterise the spatial and temporal expression patterns and subcellular localisations of PABPC1 and EPAB in vertebrate oocytes and early embryos. In the present review, we comprehensively evaluate and discuss the expression patterns and particular functions of the EPAB and PABPC1 genes, especially in mouse and human oocytes and early embryos.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.

Urb-RIP - An Adaptable and Efficient Approach for Immunoprecipitation of RNAs and Associated RNAs/Proteins.

Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb—RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the “resurrected” snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein.

Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP.

Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories.

Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.

The RNA‐binding protein LARP4 regulates cancer cell migration and invasion

LARP4 is a La‐related RNA‐binding protein implicated in regulating mRNA translation, which interacts with poly(A)‐binding protein (PABP). We previously identified LARP4 in an RNAi screen as one of several genes that regulate the shape of PC3 prostate cancer cells. Here we show that LARP4 depletion induces cell elongation in PC3 cells and MDA‐MB‐231 breast cancer cells. LARP4 depletion increases cell migration and invasion, as well as inducing invasive cell protrusions in 3D Matrigel. Conversely, LARP4 over‐expression reduces cell elongation and increases cell circularity. LARP4 mutations are found in a variety of cancers. Introduction of some of these cancer‐associated mutations, including a truncation mutant, into LARP4 enhances its effects on cell morphology. The truncation mutant shows enhanced interaction with PABP. We propose that LARP4 inhibits migration and invasion of cancer cells, and that some cancer‐associated mutations stimulate these effects of LARP4. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

PABP enhances release factor recruitment and stop codon recognition during translation termination

Poly(A)-binding protein (PABP) is a major component of the messenger RNA–protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl–tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1–eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.

Poly(A)-binding proteins are required for microRNA-mediated silencing and to promote target deadenylation in C. elegans.

Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3′ termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3′UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3′UTR poly(A) tail to the bound miRISC and deadenylase.

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

Uridylation and PABP Cooperate to Repair mRNA Deadenylated Ends in Arabidopsis.

Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis. Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis. Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A) tails. In vivo and in vitro data indicate that Poly(A) Binding Protein (PABP) binds to uridylated oligo(A) tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism.

The poly(A)-binding protein genes, EPAB, PABPC1, and PABPC3 are differentially expressed in infertile men with non-obstructive azoospermia

Azoospermia is one of the major causes of male infertility and is basically classified into obstructive (OA) and non-obstructive azoospermia (NOA). The molecular background of NOA still largely remains elusive. It has been shown that the poly(A)-binding proteins (PABPs) essentially play critical roles in stabilization and translational control of the mRNAs during spermatogenesis. In the present study, we aim to evaluate expression levels of the PABP genes, EPAB, PABPC1, and PABPC3, in the testicular biopsy samples and in the isolated spermatocyte (SC) and round spermatid (RS) fractions obtained from men with various types of NOA including hypospermatogenesis (hyposperm), RS arrest, SC arrest, and Sertoli cell-only syndrome (SCO). In the testicular biopsy samples, both PABPC1 and PABPC3 mRNA expressions were gradually decreased from hyposperm to SCO groups (P < 0.05), whereas there was no remarkable difference for the EPAB expression among groups. The expression levels of cytoplasmically localized PABPC1 and PABPC3 proteins dramatically reduced from hyposperm to SCO groups (P < 0.05). In the isolated SC and RS fractions, the EPAB, PABPC1, and PABPC3 mRNA expressions were gradually decreased from hyposperm to SC arrest groups (P < 0.05). Similarly, both PABPC1 and PABPC3 proteins were expressed at higher levels in the SC and RS fractions from hyposperm group when compared to the SC and RS fractions from either RS arrest or SC arrest group (P < 0.05). Our findings suggest that observed significant alterations in the PABPs expression may have an implication for development of different NOA forms.

MKRN1: Uncovering function by an unbiased systems approach

MKRN1: Uncovering function by an unbiased systems approach

Poly(A)-binding proteins and mRNA localization: who rules the roost?

RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals.

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity.

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein.

Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.