poly(A)-binding Protein Research Articles

Polyadenylation of Sine Transcripts Generated by RNA Polymerase III Dramatically Prolongs Their Lifetime in Cells

Short Interspersed Elements (SINEs) are mobile genetic elements of higher eukaryotes, which originated during evolution from various tRNAs and less often from 5S rRNA and 7SL RNA. Similar to the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have an ability to undergo AAUAAA-dependent polyadenylation, which is unique for the RNA polymerase III transcripts. It is well known that this polyadenylation of many RNA polymerase II transcripts (e.g., mRNAs) increases their lifetime in the cell. The aim of this work is to examine whether the stability of SINE transcripts increases as a result of AAUAAA-dependent polyadenylation. HeLa cells were transfected with SINE DNA, both containing and not containing the polyadenylation signal (AATAAA). One day later, the transcription was inhibited by actinomycin D, and the decrease in the level of the SINE transcripts was monitored by northern hybridization. For all the eight studied SINEs, the half-life of nonpolyadenylated transcripts was 20-30 minutes, and for polyadenylated transcripts, this parameter exceeded 3 hours. Interestingly, the insertion of an additional 80-bp DNA fragment into the middle region of B2 SINE did not significantly reduce the stability of the polyadenylated transcripts. It is most likely that the increase in the lifetime of the polyadenylated SINE transcripts is due to the fact that the poly(A) tail interacts with the poly(A)-binding proteins (PABPs), thus protecting the RNA from degradation by the exonucleases acting from the 3'-end. The results make it possible to design SINE-based vectors intended for the expression of short noncoding RNAs, which are stable in a cell due to polyadenylation.

Genomic survey of RNA recognition motif (RRM) containing RNA binding proteins from barley (Hordeum vulgare ssp. vulgare)

One of the major mechanisms of post-transcriptional gene regulation is achieved by proteins bearing well-defined sequence motifs involved in ‘RNA binding’. In eukaryotes, RNA binding proteins (RBPs) are key players of RNA metabolism that includes synthesis, processing, editing, modifying, transport, storage and stability of RNA. In plants, the family of RBPs is vastly expanded compared to other eukaryotes including humans. In this study we identified 363 RBPs in the barley genome. Gene ontology enrichment analysis of barley RBPs indicated these proteins were in all the major cellular compartments and associated with key biological processes including translation, splicing, seed development and stress signaling. Members with the classical RNA binding motifs such as the RNA recognition motif (RRM), KH domain, Helicase, CRM, dsRNA and Pumilio were identified in the repertoire of barley RBPs. Similar to Arabidopsis, the RRM containing RBPs were the most abundant in barley genome. In-depth analysis of the RRM containing proteins - polyA binding proteins, Ser/Arg rich proteins and Glycine-rich RBPs were undertaken. Reanalysis of the proteome dataset of various stages during barley malting identified 38 RBPs suggesting an important role for these proteins during the malting process. This survey provides a systematic analysis of barley RBPs and serves as the basis for the further functional characterization of this important family of proteins.

The Interactome analysis of the Respiratory Syncytial Virus protein M2-1 suggests a new role in viral mRNA metabolism post-transcription

Human respiratory syncytial virus (RSV) is a globally prevalent negative-stranded RNA virus, which can cause life-threatening respiratory infections in young children, elderly people and immunocompromised patients. Its transcription termination factor M2-1 plays an essential role in viral transcription, but the mechanisms underpinning its function are still unclear. We investigated the cellular interactome of M2-1 using green fluorescent protein (GFP)-trap immunoprecipitation on RSV infected cells coupled with mass spectrometry analysis. We identified 137 potential cellular partners of M2-1, among which many proteins associated with mRNA metabolism, and particularly mRNA maturation, translation and stabilization. Among these, the cytoplasmic polyA-binding protein 1 (PABPC1), a candidate with a major role in both translation and mRNA stabilization, was confirmed to interact with M2-1 using protein complementation assay and specific immunoprecipitation. PABPC1 was also shown to colocalize with M2-1 from its accumulation in inclusion bodies associated granules (IBAGs) to its liberation in the cytoplasm. Altogether, these results strongly suggest that M2-1 interacts with viral mRNA and mRNA metabolism factors from transcription to translation, and imply that M2-1 may have an additional role in the fate of viral mRNA downstream of transcription.

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence' and `replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence' and `replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.

LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA.

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A–oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein–protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.

Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei

In Trypanosoma brucei, most mitochondrial mRNAs undergo editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for translation. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes mRNA degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point likely prevents translation of incompletely edited mRNAs. We also find that RNA editing substrate binding complex (RESC) mediates the interaction between the 5′ end-bound pyrophosphohydrolase MERS1 and 3′ end-associated KPAF4 to enable mRNA circularization. This event appears to be critical for edited mRNA stability.

Escaping nuclear decay: the significance of mRNA export for gene expression.

In this perspective, we discuss the regulatory impact of nuclear RNA export and decay on messenger RNA (mRNA) functionality. It is well established that control of protein-coding gene expression in eukaryotes employs the regulated production of mRNA, its intra-cellular transfer to cytoplasmic ribosomes and final transcript degradation. Despite a rich body of literature on these events, an involvement of nuclear RNA decay systems remains largely unexplored. Instead, nuclear RNA degradation is often considered a quality control precaution engaged primarily in ridding cells of aberrantly processed transcripts and spurious non-coding RNA. Recent research from human and budding yeast cells, however, demonstrates that even protein-coding transcripts fall prey to nuclear decay and that this is countered by their nuclear export. Here, we outline the potential of nuclear polyA-binding proteins in tuning levels of cellular mRNA to maintain transcript homeostasis.

Tales of Detailed Poly(A) Tails.

Poly(A) tails are non-templated additions of adenosines at the 3' ends of most eukaryotic mRNAs. In the nucleus, these RNAs are co-transcriptionally cleaved at a poly(A) site and then polyadenylated before being exported to the cytoplasm. In the cytoplasm, poly(A) tails play pivotal roles in the translation and stability of the mRNA. One challenge in studying poly(A) tails is that they are difficult to sequence and accurately measure. However, recent advances in sequencing technology, computational algorithms, and other assays have enabled a more detailed look at poly(A) tail length genome-wide throughout many developmental stages and organisms. With the help of these advances, our understanding of poly(A) tail length has evolved over the past 5 years with the recognition that highly expressed genes can have short poly(A) tails and the elucidation of the seemingly contradictory roles for poly(A)-binding protein (PABP) in facilitating both protection and deadenylation.

Dynamic interaction of poly(A)-binding protein with the ribosome

Eukaryotic mRNA has a cap structure and a poly(A) tail at the 5′ and 3′ ends, respectively. The cap structure is recognized by eIF (eukaryotic translation initiation factor) 4 F, while the poly(A) tail is bound by poly(A)-binding protein (PABP). PABP has four RNA recognition motifs (RRM1–4), and RRM1-2 binds both the poly(A) tail and eIF4G component of eIF4F, resulting in enhancement of translation. Here, we show that PABP interacts with the 40S and 60S ribosomal subunits dynamically via RRM2-3 or RRM3-4. Using a reconstituted protein expression system, we demonstrate that wild-type PABP activates translation in a dose-dependent manner, while a PABP mutant that binds poly(A) RNA and eIF4G, but not the ribosome, fails to do so. From these results, functional significance of the interaction of PABP with the ribosome is discussed.

Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell.

In the present study, we investigated the roles of interactions among the poly(A) tail, coronavirus nucleocapsid (N) protein, and poly(A)-binding protein (PABP) in the regulation of coronavirus gene expression. Through dissociation constant (Kd ) comparison, we found that the coronavirus N protein can bind to the poly(A) tail with high affinity, establishing N protein as a PABP. A subsequent analysis with UV cross-linking and immunoprecipitation revealed that the N protein is able to bind to the poly(A) tail in infected cells. Further examination demonstrated that poly(A) tail binding by the N protein negatively regulates translation of coronaviral RNA and host mRNA both in vitro and in cells. Although the N protein can interact with PABP and eukaryotic initiation factor 4G (eIF4G), the poor interaction efficiency between the poly(A)-bound N protein and eIF4E may explain the observed decreased translation efficiency. In addition to interaction with translation factor eIF4G, the N protein is able to interact with coronavirus nonstructural protein 9 (nsp9), a replicase protein required for replication. The study demonstrates interactions among the poly(A) tail, N protein, and PABP both in vitro and in infected cells. Of the interactions, binding of the poly(A) tail to N protein decreases the interaction efficiency between the poly(A) tail and eIF4E, leading to translation inhibition. The poly(A)-dependent translation inhibition by N protein has not been previously demonstrated and thus extends our understanding of coronavirus gene expression.IMPORTANCE Gene expression in coronavirus is a complicated and dynamic process. In this study, we demonstrated that coronavirus N protein is able to bind to the poly(A) tail with high affinity, establishing N protein as a PABP. We also show how the interplay between coronavirus 3' poly(A) tail, PABP, and N protein regulates gene expression of the coronavirus and host cell. Of the interactions, poly(A) tail binding by the N protein negatively regulates translation, and to our knowledge, this inhibition of translation by binding of the N protein to poly(A) tail has not been previously studied. Accordingly, the study provides fundamental molecular details regarding coronavirus infection and expands our knowledge of coronavirus gene expression.

The zinc finger protein DCM1 is required for male meiotic cytokinesis by preserving callose in rice.

Meiotic cytokinesis influences the fertility and ploidy of gametes. However, limited information is available on the genetic control of meiotic cytokinesis in plants. Here, we identified a rice mutant with low male fertility, defective callose in meiosis 1 (dcm1). The pollen grains of dcm1 are proved to be defective in exine formation. Meiotic cytokinesis is disrupted in dcm1, resulting in disordered spindle orientation during meiosis II and formation of pollen grains with varied size and DNA content. We demonstrated that meiotic cytokinesis defect in dcm1 is caused by prematurely dissolution of callosic plates. Furthermore, peripheral callose surrounding the dcm1 pollen mother cells (PMCs) also disappeared untimely around pachytene. The DCM1 protein contains five tandem CCCH motifs and interacts with nuclear poly (A) binding proteins (PABNs) in nuclear speckles. The expression profiles of genes related to callose synthesis and degradation are significantly modified in dcm1. Together, we propose that DCM1 plays an essential role in male meiotic cytokinesis by preserving callose from prematurely dissolution in rice.

Shep interacts with posttranscriptional regulators to control dendrite morphogenesis in sensory neurons

RNA binding proteins (RBPs) mediate posttranscriptional gene regulatory events throughout development. During neurogenesis, many RBPs are required for proper dendrite morphogenesis within Drosophila sensory neurons. Despite their fundamental role in neuronal morphogenesis, little is known about the molecular mechanisms in which most RBPs participate during neurogenesis. In Drosophila, alan shepard (shep) encodes a highly conserved RBP that regulates dendrite morphogenesis in sensory neurons. Moreover, the C. elegans ortholog sup-26 has also been implicated in sensory neuron dendrite morphogenesis. Nonetheless, the molecular mechanism by which Shep/SUP-26 regulate dendrite development is not understood. Here we show that Shep interacts with the RBPs Trailer Hitch (Tral), Ypsilon schachtel (Yps), Belle (Bel), and Poly(A)-Binding Protein (PABP), to direct dendrite morphogenesis in Drosophila sensory neurons. Moreover, we identify a conserved set of Shep/SUP-26 target RNAs that include regulators of cell signaling, posttranscriptional gene regulators, and known regulators of dendrite development.

Multiple Posttranscriptional Strategies To Regulate the Herpes Simplex Virus 1 vhs Endoribonuclease.

The herpes simplex virus 1 (HSV-1) virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in the shutoff of host protein synthesis. Hence, its unrestrained activity is considered lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with the aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from cotransfected plasmids were also retained in the same nuclei where vhs mRNA was located, while poly(A) binding protein (PABP) was relocalized to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Coexpression of VP16 and VP22 rescued the cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous green fluorescent protein transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and autoinduced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV-1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs but that must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted posttranscriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and coexpressed mRNAs for nuclear retention, an activity that is relieved by coexpression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors coordinate gene expression at the time that they are needed. These findings are broadly relevant to both virus and cellular gene expression.

Regulación de la traducción mediada por la proteína NSP3 de rotavirus

El estudio de los mecanismos utilizados por diversos virus para controlar la traducción de la célula hospedera y favorecer la expresión de proteínas virales ha sido muy instructivo para conocer mecanismos celulares fundamentales que regulan la traducción. La proteína NSP3 de los rotavirus de la especie A (RVA) es capaz de inhibir la traducción de los RNAm celulares como consecuencia de su unión al factor de iniciación de la traducción eIF4G y de estimular la traducción de los RNAm virales como consecuencia de su unión a la secuencia UGACC de su extremo 3’. Se ha propuesto que NSP3 inhibe la traducción por interferir con la circularización de los extremos 5’-3’ de los RNAm celulares mediada por eIF4E-eIF4G-PABP (proteína de unión a poli-A), y simultáneamente se propone que NSP3 estimula la traducción de los RNAm del RVA circularizándolos de manera análoga a la que realiza PABP en los RNAm celulares. Sin embargo, la importancia de la dimerización de NSP3 asistida por la chaperona HSP90 en su función inhibitoria de la traducción celular se desconoce. Recientemente, se exploró la importancia de los intermediarios de la dimerización de NSP3 sobre su función inhibitoria de la traducción, y se encontró que mutaciones puntuales en la región coiled-coil afectan la formación de los dímeros y conservan parcialmente su función inhibitoria de la traducción celular. Además, se detectó que los dímeros de NSP3 se degradan con mayor rapidez que los intermediarios de la dimerización. Estos datos demuestran que la función de NSP3 se adquiere previamente a la aparición de los dímeros, y sugiere que la susceptibilidad al proteasoma de las distintas formas oligoméricas de NSP3 son relevantes en el establecimiento de la función inhibitoria de la traducción celular.

PABP Cooperates with the CCR4-NOT Complex to Promote mRNA Deadenylation and Block Precocious Decay

Multiple deadenylases are known in vertebrates, thePAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>∼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the ∼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.

RNA Modulates the Interaction between Influenza A Virus NS1 and Human PABP1.

Nonstructural protein 1 (NS1) is a multifunctional protein involved in preventing host-interferon response in influenza A virus (IAV). Previous studies have indicated that NS1 also stimulates the translation of viral mRNA by binding to conserved sequences in the viral 5'-UTR. Additionally, NS1 binds to poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G (eIF4G). The interaction of NS1 with the viral 5'-UTR, PABP1, and eIF4G has been suggested to specifically enhance the translation of viral mRNAs. In contrast, we report that NS1 does not directly bind to sequences in the viral 5'-UTR, indicating that NS1 is not responsible for providing the specificity to stimulate viral mRNA translation. We also monitored the interaction of NS1 with PABP1 using a new, quantitative FRET assay. Our data show that NS1 binds to PABP1 with high affinity; however, the binding of double-stranded RNA (dsRNA) to NS1 weakens the binding of NS1 to PABP1. Correspondingly, the binding of PABP1 to NS1 weakens the binding of NS1 to double-stranded RNA (dsRNA). In contrast, the affinity of PABP1 for binding to poly(A) RNA is not significantly changed by NS1. We propose that the modulation of NS1·PABP1 interaction by dsRNA may be important for the viral cycle.

Domain-functional analyses of PIWIL1 and PABPC1 indicate their synergistic roles in protein translation via 3'-UTRs of meiotic mRNAs.

Translational regulation plays a central role during post-meiotic development of male germ cells. Previous studies suggested that P-element induced wimpy testis like 1 (PIWIL1), a PIWI-interacting RNA (piRNA) binding protein that is critical for sperm development, participates in the maintenance and translational regulation of post-meiotic mRNAs in haploid spermatids. However, how PIWIL1 regulates protein translation remains largely unclear. Using biochemical assays, we show here that PIWIL1 utilizes different domains to interact with post-meiotic mRNAs and Poly-A binding protein cytoplasmic 1 (PABPC1), a general protein translation regulator. PIWIL1 binds 3'-untranslated regions (3'-UTRs) of several spermiogenic mRNAs via its N-terminal domain, whereas its interactions with PABPC1 are mediated through its N- and C-terminal domains in an RNA-dependent manner. Using a heterologous cell system, we analyzed its effects on protein translation via luciferase reporter assay and sucrose gradient sedimentation. It was found that PIWIL1 augments protein translation with PABPC1 in the presence of 3'-UTRs of post-meiotic mRNAs. While both the N-terminal RNA recognition motif (RRM) domain and the central linker region of PABPC1 stimulate translation, only the PIWI Argonaute and Zwille (PAZ) domain of PIWIL1 positively affects translation of reporter mRNAs. Interestingly, the PAZ domain was found absent from polysomal fractions, in contrast to the N- and C-terminal domains of PIWIL1. Taken together, the results suggest that PIWIL1 interacts with various partners using different domains and participates in translational regulation partly through 3'-UTRs. It will be of interest to further explore how PIWIL1 elicit its versatile functions, including translational regulation of post-meiotic mRNAs through intrinsic structural changes and extrinsic signals during mouse spermiogenesis under more physiological settings.

Toxoplasma gondii infection induces the formation of host's nuclear granules containing poly(A)-binding proteins.

To study the mechanism by which human host cells respond to an infection of Toxoplasma gondii, we monitored the level of poly(A)-binding protein (PABP), an indicator of translation. Here, we report an observation of the relocalization of PABPs in human host cells upon T. gondii infection. Notably, the aggregates of PABPs formed upon infection are mainly found in the nucleus, which is a different response from that found after exposure to heat shock. Pyrimethamine treatment of the infected monolayers inhibits the multiplicity of the parasite and reverses the relocalization of PABP aggregates. This active interaction between the infected mammalian host cells and T. gondii appears to be different from that caused by viral infection.

MicroRNP‐mediated translational activation of nonadenylated mRNAs in a mammalian cell‐free system

MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4-NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let-7-microRNP-mediated translational activation of nonadenylated target mRNAs in a mammalian cell-free system, which contains over-expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4-1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)-binding protein (PABP) interacting motif (PAM2) from the TNRC6B-SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP-mediated mRNA regulation.