miRNA-mediated Translational Repression Research Articles

Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

An essential role for the RNA helicase DDX6 in NMDA receptor-dependent gene silencing and dendritic spine shrinkage

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins in the RNA-induced silencing complex (RISC) to modulate protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by repressing the translation of proteins involved in dendritic spine morphogenesis. Rapid NMDAR-dependent silencing of Limk1 is essential for spine shrinkage and requires Ago2 phosphorylation at S387. Not all gene silencing events are modulated by S387 phosphorylation, and the mechanisms that govern the selection of specific mRNAs for silencing downstream of S387 phosphorylation are unknown. Here, we show that NMDAR-dependent S387 phosphorylation causes a rapid and transient increase in the association of Ago2 with Limk1, but not Apt1 mRNA. The specific increase in Limk1 mRNA binding to Ago2 requires recruitment of the helicase DDX6 to RISC. Furthermore, we show that DDX6 is required for NMDAR-dependent silencing of Limk1 via miR-134, but not Apt1 via miR-138, and is essential for NMDAR-dependent spine shrinkage. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression of specific genes to control dendritic spine morphology.

SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression.

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5´-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-ß, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) via GIGYF2 and enhances the translational repression mediated by natural miRNA binding sites in the 3´ UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery.

A Fast-Track Phenotypic Characterization of Plasmodium falciparum Vaccine Antigens through Lyse-Reseal Erythrocytes Mediated Delivery (LyRED) of RNA Interference for Targeted Translational Repression.

The minimal success of the malaria vaccine with available antigens indicates the need for intensive and accelerated research to identify and characterize new antigens that confer protection against infection, clinical manifestation, and even malaria transmission. Further, the genetic manipulation tools to characterize such antigens are very time-consuming and laborious due to the very low efficiency of transfection in the malaria parasite. Here, we report a human miRNA-mediated translational repression of antigens in Plasmodium falciparum as a fast-track method for understanding and validating their function. In this method, candidate miRNAs are designed based on favorable hybridization energy against a parasite gene, and miRNA mimics are delivered to the parasite by loading them as cargo in the erythrocytes by simple lyse-reseal method. Incubation of the miRNA loaded erythrocytes with purified mature trophozoites or schizonts results in the loaded erythrocytes' infection. The miRNA mimics are translocated to parasites, and the effect of miRNA-mediated translation repression can be monitored within 48-72h post-invasion. Unlike other transfection based methods, this method is fast, reproducible, and robust. We call this method as lyse-reseal erythrocytes for delivery (LyRED) of miRNA, which is a rapid and straight-forward method providing an efficient alternative to the existing genetic tools for P. falciparum to characterize the function of antigens or genes. The identification of crucial antigens from the different stages of the Plasmodium falciparum life cycle by the miRNA targeting approach can fuel the development of efficacious subunit vaccines against malaria.

LncRNA-ATB regulates epithelial-mesenchymal transition progression in pulmonary fibrosis via sponging miR-29b-2-5p and miR-34c-3p.

Dysregulation of non‐coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial‐mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non‐coding RNA (lncRNA) ATB in silica‐induced pulmonary fibrosis‐related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR‐29b‐2‐5p and miR‐34c‐3p as two vital downstream targets of lncRNA‐ATB. As opposed to lncRNA‐ATB, a significant reduction of both miR‐29b‐2‐5p and miR‐34c‐3p was observed in lung epithelial cells treated with TGF‐β1 and a murine silicosis model. Overexpression miR‐29b‐2‐5p or miR‐34c‐3p inhibited EMT process and abrogated the pro‐fibrotic effects of lncRNA‐ATB in vitro. Further, the ectopic expression of miR‐29b‐2‐5p and miR‐34c‐3p with chemotherapy attenuated silica‐induced pulmonary fibrosis in vivo. Mechanistically, TGF‐β1‐induced lncRNA‐ATB accelerated EMT as a sponge of miR‐29b‐2‐5p and miR‐34c‐3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA‐mediated translational repression. Interestingly, the co‐transfection of miR‐29b‐2‐5p and miR‐34c‐3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co‐expression of these two miRNAs by using adeno‐associated virus (AAV) better alleviated silica‐induced fibrogenesis than single miRNA. Approaches aiming at lncRNA‐ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis.

Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum.

Translational repression is a conserved mechanism in microRNA (miRNA)-guided gene silencing. In Arabidopsis, ARGONAUTE1 (AGO1), the major miRNA effector, localizes in the cytoplasm for mRNA cleavage and at the endoplasmic reticulum (ER) for translational repression of target genes. However, the mechanism underlying miRNA-mediated translational repression is poorly understood. In particular, how the subcellular partitioning of AGO1 is regulated is largely unexplored. Here, we show that the plant hormone brassinosteroids (BRs) inhibit miRNA-mediated translational repression by negatively regulating the distribution of AGO1 at the ER in Arabidopsis thaliana. We show that the protein levels rather than the transcript levels of miRNA target genes were reduced in BR-deficient mutants but increased under BR treatments. The localization of AGO1 at the ER was significantly decreased under BR treatments while it was increased in the BR-deficient mutants. Moreover, ROTUNDIFOLIA3 (ROT3), an enzyme involved in BR biosynthesis, co-localizes with AGO1 at the ER and interacts with AGO1 in a GW motif-dependent manner. Complementation analysis showed that the AGO1-ROT3 interaction is necessary for the function of ROT3. Our findings provide new clues to understand how miRNA-mediated gene silencing is regulated by plant endogenous hormones.

Cytoplasmic HYL1 modulates miRNA-mediated translational repression.

MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation.

MicroRNA-mediated translation repression through GYF-1 and IFE-4 in C. elegans development.

microRNA (miRNA)-mediated gene silencing is enacted through the recruitment of effector proteins that direct translational repression or degradation of mRNA targets, but the relative importance of their activities for animal development remains unknown. Our concerted proteomic surveys identified the uncharacterized GYF-domain encoding protein GYF-1 and its direct interaction with IFE-4, the ortholog of the mammalian translation repressor 4EHP, as key miRNA effector proteins in Caenorhabditis elegans. Recruitment of GYF-1 protein to mRNA reporters in vitro or in vivo leads to potent translation repression without affecting the poly(A) tail or impinging on mRNA stability. Loss of gyf-1 is synthetic lethal with hypomorphic alleles of embryonic miR-35–42 and larval (L4) let-7 miRNAs, which is phenocopied through engineered mutations in gyf-1 that abolish interaction with IFE-4. GYF-1/4EHP function is cascade-specific, as loss of gyf-1 had no noticeable impact on the functions of other miRNAs, including lin-4 and lsy-6. Overall, our findings reveal the first direct effector of miRNA-mediated translational repression in C. elegans and its physiological importance for the function of several, but likely not all miRNAs.

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4+, CD8+ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3+ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.

ALTERED MERISTEM PROGRAM1 regulates leaf identity independently of miR156-mediated translational repression.

In Arabidopsis, loss of the carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) produces an increase in the rate of leaf initiation, an enlarged shoot apical meristem and an increase in the number of juvenile leaves. This phenotype is also observed in plants with reduced levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, suggesting that AMP1 might promote SPL activity. However, we found that the amp1 mutant phenotype is only partially corrected by elevated SPL gene expression, and that amp1 has no significant effect on SPL transcript levels, or on the level or the activity of miR156. Although AMP1 has been reported to promote miRNA-mediated translational repression, amp1 did not prevent the translational repression of the miR156 target SPL9 or the miR159 target MYB33. These results suggest that AMP1 regulates vegetative phase change downstream of, or in parallel to, the miR156/SPL pathway, and that it is not universally required for miRNA-mediated translational repression.

MLL is required for miRNA-mediated translational repression

MLL is required for miRNA-mediated translational repression

Waking the sleeping dragon: gene expression profiling reveals adaptive strategies of the hibernating reptile Pogona vitticeps

BackgroundHibernation is a physiological state exploited by many animals exposed to prolonged adverse environmental conditions associated with winter. Large changes in metabolism and cellular function occur, with many stress response pathways modulated to tolerate physiological challenges that might otherwise be lethal. Many studies have sought to elucidate the molecular mechanisms of mammalian hibernation, but detailed analyses are lacking in reptiles. Here we examine gene expression in the Australian central bearded dragon (Pogona vitticeps) using mRNA-seq and label-free quantitative mass spectrometry in matched brain, heart and skeletal muscle samples from animals at late hibernation, 2 days post-arousal and 2 months post-arousal.ResultsWe identified differentially expressed genes in all tissues between hibernation and post-arousal time points; with 4264 differentially expressed genes in brain, 5340 differentially expressed genes in heart, and 5587 differentially expressed genes in skeletal muscle. Furthermore, we identified 2482 differentially expressed genes across all tissues. Proteomic analysis identified 743 proteins (58 differentially expressed) in brain, 535 (57 differentially expressed) in heart, and 337 (36 differentially expressed) in skeletal muscle. Tissue-specific analyses revealed enrichment of protective mechanisms in all tissues, including neuroprotective pathways in brain, cardiac hypertrophic processes in heart, and atrophy protective pathways in skeletal muscle. In all tissues stress response pathways were induced during hibernation, as well as evidence for gene expression regulation at transcription, translation and post-translation.ConclusionsThese results reveal critical stress response pathways and protective mechanisms that allow for maintenance of both tissue-specific function, and survival during hibernation in the central bearded dragon. Furthermore, we provide evidence for multiple levels of gene expression regulation during hibernation, particularly enrichment of miRNA-mediated translational repression machinery; a process that would allow for rapid and energy efficient reactivation of translation from mature mRNA molecules at arousal. This study is the first molecular investigation of its kind in a hibernating reptile, and identifies strategies not yet observed in other hibernators to cope stress associated with this remarkable state of metabolic depression.

Extensive profiling in Arabidopsis reveals abundant polysome-associated 24-nt small RNAs including AGO5-dependent pseudogene-derived siRNAs.

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In Arabidopsis, this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action. Using AGO1:miRNA-mediated TR as a paradigm, we explored, with two unrelated polysome-isolation methods, which, among the ten Arabidopsis AGOs and numerous sRNA classes, interact with translation. We found that representatives of all three AGO-clades associate with polysomes, including the TGS-effector AGO4 and stereotypical 24-nt sRNAs that normally mediate TGS of transposons/repeats. Strikingly, approximately half of these annotated 24-nt siRNAs displayed unique matches in coding regions/introns of genes, and in pseudogenes, but not in transposons/repeats commonly found in their vicinity. Protein-coding gene-derived 24-nt sRNAs correlate with gene-body methylation. Those derived from pseudogenes belong to two main clusters defined by their parental-gene expression patterns, and are vastly enriched in AGO5, itself found on polysomes. Based on their tight expression pattern in developing and mature siliques, their biogenesis, and genomic/epigenomic features of their loci-of-origin, we discuss potential roles for these hitherto unknown polysome-enriched, pseudogene-derived siRNAs.

ALTERED MERISTEM PROGRAM1 Restricts Shoot Meristem Proliferation and Regeneration by Limiting HD-ZIP III-Mediated Expression of RAP2.6L.

Plants show an indeterminate mode of growth by the activity of organ forming stem cell niches in apically positioned meristems. The correct formation and activity of these meristems are a prerequisite for their adaptive development and also allow the maintenance of organogenesis under adverse circumstances such as wounding. Mutation of the putative Arabidopsis (Arabidopsis thaliana) Glu carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) results in Arabidopsis plants with enlarged shoot apical meristems, supernumerary stem cell pools, and higher leaf formation rate. AMP1 deficiency also causes exaggerated de novo formation of shoot meristems. The activity of AMP1 has been implicated in the control of microRNA (miRNA)-dependent translation; however, it is not known how this function contributes to the shoot meristem defects. Here, we show that the transcription factor RAP2.6L is upregulated in the Arabidopsis amp1 mutant. Overexpression of RAP2.6L in the wild type causes amp1 mutant-related phenotypic and molecular defects, including enhanced shoot regeneration in tissue culture. Conversely, inhibition of RAP2.6L in the amp1 mutant suppresses stem cell hypertrophy and the regenerative capacity. We further provide evidence that RAP2.6L is under direct transcriptional control of miRNA-regulated class III homeodomain-Leu zipper (HD-ZIP III) proteins, key regulators of shoot meristem development, which overaccumulate in the amp1 mutant. Our results reveal a transcription factor module acting downstream of AMP1 in the control of shoot stem cell niche patterning. By positioning the HD-ZIP III/RAP2.6L module downstream of AMP1 function, we provide a mechanistic link between the role of AMP1 in miRNA-mediated translational repression and shoot stem cell specification.

NMDAR-dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity.

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.

Translation efficiency is a determinant of the magnitude of miRNA-mediated repression

MicroRNAs are well known regulators of mRNA stability and translation. However, the magnitude of both translational repression and mRNA decay induced by miRNA binding varies greatly between miRNA targets. This can be the result of cis and trans factors that affect miRNA binding or action. We set out to address this issue by studying how various mRNA characteristics affect miRNA-mediated repression. Using a dual luciferase reporter system, we systematically analyzed the ability of selected mRNA elements to modulate miRNA-mediated repression. We found that changing the 3′UTR of a miRNA-targeted reporter modulates translational repression by affecting the translation efficiency. This 3′UTR dependent modulation can be further altered by changing the codon-optimality or 5′UTR of the luciferase reporter. We observed maximal repression with intermediate codon optimality and weak repression with very high or low codon optimality. Analysis of ribosome profiling and RNA-seq data for endogenous miRNA targets revealed translation efficiency as a key determinant of the magnitude of miRNA-mediated translational repression. Messages with high translation efficiency were more robustly repressed. Together our results reveal modulation of miRNA-mediated repression by characteristics and features of the 5′UTR, CDS and 3′UTR.

The PICK1 Ca2+ sensor modulates N-methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons

MicroRNAs (miRNAs) are important regulators of localized mRNA translation in neuronal dendrites. The presence of RNA-induced silencing complex proteins in these compartments and the dynamic miRNA expression changes that occur in response to neuronal stimulation highlight their importance in synaptic plasticity. Previously, we demonstrated a novel interaction between the major RNA-induced silencing complex component Argounaute-2 (Ago2) and the BAR (bin/amphiphysin/rvs) domain protein PICK1. PICK1 recruits Ago2 to recycling endosomes in dendrites, where it inhibits miRNA-mediated translational repression. Chemical induction of long-term depression via NMDA receptor activation causes the dissociation of Ago2 from PICK1 and a consequent increase in dendritic miRNA-mediated gene silencing. The mechanism that underlies the regulation of PICK1-Ago2 binding is unknown. In this study, we demonstrate that the PICK1-Ago2 interaction is directly sensitive to Ca2+ ions so that high [Ca2+]free reduces PICK1 binding to Ago2. Mutating a stretch of C-terminal Ca2+-binding residues in PICK1 results in a complete block of NMDA-induced PICK1-Ago2 disassociation in cortical neurons. Furthermore, the same mutant also blocks NMDA-stimulated miRNA-mediated gene silencing. This study defines a novel mechanism whereby elevated [Ca2+] induced by NMDA receptor activation modulates Ago2 and miRNA activity via PICK1. Our work suggests a Ca2+-dependent process to regulate miRNA activity in neurons in response to the induction of long-term depression.

The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. For example, miRNAs repress gene expression by recruiting the miRNA-induced silencing complex (miRISC), a ribonucleoprotein complex that contains miRNA-engaged Argonaute (Ago) and the scaffold protein GW182. Recently, ubiquitin-protein ligase E3 component N-recognin 5 (UBR5) has been identified as a component of miRISC. UBR5 directly interacts with GW182 proteins and participates in miRNA silencing by recruiting downstream effectors, such as the translation regulator DEAD-box helicase 6 (DDX6) and transducer of ERBB2,1/2,2 (Tob1/2), to the Ago-GW182 complex. However, the regulation of miRISC-associated UBR5 remains largely elusive. In the present study, we showed that UBR5 down-regulates the levels of TNF receptor-associated factor 3 (TRAF3), a key component of Toll-like receptor signaling, via the miRNA pathway. We further demonstrated that p90 ribosomal S6 kinase (p90RSK) is an upstream regulator of UBR5. p90RSK phosphorylates UBR5 at Thr637, Ser1227, and Ser2483, and this phosphorylation is required for the translational repression of TRAF3 mRNA. Phosphorylated UBR5 co-localized with GW182 and Ago2 in cytoplasmic speckles, which implies that miRISC is affected by phospho-UBR5. Collectively, these results indicated that the p90RSK-UBR5 pathway stimulates miRNA-mediated translational repression of TRAF3. Our work has added another layer to the regulation of miRISC.

Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein–protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.

Activity-dependent spatially localized miRNA maturation in neuronal dendrites.

MicroRNAs (miRNAs) regulate gene expression by binding to target messenger RNAs (mRNAs) and preventing their translation. In general, the number of potential mRNA targets in a cell is much greater than the miRNA copy number, complicating high-fidelity miRNA-target interactions. We developed an inducible fluorescent probe to explore whether the maturation of a miRNA could be regulated in space and time in neurons. A precursor miRNA (pre-miRNA) probe exhibited an activity-dependent increase in fluorescence, suggesting the stimulation of miRNA maturation. Single-synapse stimulation resulted in a local maturation of miRNA that was associated with a spatially restricted reduction in the protein synthesis of a target mRNA. Thus, the spatially and temporally regulated maturation of pre-miRNAs can be used to increase the precision and robustness of miRNA-mediated translational repression.