Regulation Of microRNA Biogenesis Research Articles

MIR194-2HG, a miRNA host gene activated by HNF4A, inhibits gastric cancer by regulating microRNA biogenesis

BackgroundMicroRNA host gene (MIRHG) lncRNA is a particular lncRNA subclass that can perform both typical and atypical lncRNA functions. The biological function of MIRHG lncRNA MIR194-2HG in cancer is poorly understood.MethodsLoss-of-function studies were performed in vivo and in vitro to reveal the biological function of MIR194-2HG in GC. MicroRNA PCR array, northern blotting, RNA sequencing, chromatin immunoprecipitation, and rescue assays were conducted to uncover the molecular mechanism of MIR194-2HG.ResultsIn this study, we reported an atypical lncRNA function of MIR194-2HG in GC. MIR194-2HG downregulation was clinically associated with malignant progression and poor prognosis in GC. Functional assays confirmed that MIR194-2HG knockdown significantly promoted GC proliferation and metastasis in vitro and in vivo. Mechanismically, MIR194-2HG was required for the biogenesis of miR-194 and miR-192, which were reported to be tumor-suppressor genes in GC. Moreover, hepatocyte nuclear factor HNF4A directly activated the transcription of MIR194-2HG and its derived miR-194 and miR-192. Meanwhile, BTF3L4 was proved to be a common target gene of miR-192 and miR-194. Rescue assay further confirmed that MIR194-2HG knockdown promotes GC progression through maintaining BTF3L4 overexpression in a miR-194/192-dependent manner.ConclusionThe dysregulated MIR194-2HG/BTF3L4 axis is responsible for GC progression. Targeting HNF4A to inhibit miR-192/194 expression may be a promising strategy for overcoming GC.

PLK1 Regulates MicroRNA Biogenesis through Drosha Phosphorylation.

Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3β, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.

Environmental-relevant bisphenol A exposure promotes ovarian cancer stemness by regulating microRNA biogenesis.

Bisphenol A (BPA) is a ubiquitous environmental xenobiotic impacting millions of people worldwide. BPA has long been proposed to promote ovarian carcinogenesis, but the detrimental mechanistic target remains unclear. Cancer stem cells (CSCs) are considered as the trigger of tumour initiation and progression. Here, we show for the first time that nanomolar (environmentally relevant) concentration of BPA can markedly increase the formation and expansion of ovarian CSCs concomitant. This effect is observed in both oestrogen receptor (ER)-positive and ER-defective ovarian cancer cells, suggesting that is independent of the classical ERs. Rather, the signal is mediated through alternative ER G-protein-coupled receptor 30 (GPR30), but not oestrogen-related receptor α and γ. Moreover, we report a novel role of BPA in the regulation of Exportin-5 that led to dysregulation of microRNA biogenesis through miR-21. The use of GPR30 siRNA or antagonist to inhibit GPR30 expression or activity, respectively, resulted in significant inhibition of ovarian CSCs. Similarly, the CSCs phenotype can be reversed by expression of Exportin-5 siRNA. These results identify for the first time non-classical ER and microRNA dysregulation as novel mediators of low, physiological levels of BPA function in CSCs that may underlie its significant tumour-promoting properties in ovarian cancer.

Abstract 86: Dicer Ablation In Oligodendrocytes Impairs Oligodendrogenesis And Functional Recovery After Stroke

Introduction: Stroke-induced oligodendrogenesis contributes to ischemic brain remodeling and spontaneous functional recovery. Genes that regulate microRNA biogenesis in stroke-induced oligodendrogenesis remain little known. Methods: We employed a transgenic mouse line with conditional and inducible ablation of Dicer, a key gene of miRNA biogenesis, in myelin proteolipid protein (PLP) lineage oligodendrocytes (Dicer-KO). A PLP reporter mouse line was used as a control. Mice were subjected to transient (60min) middle cerebral artery occlusion (MCAO, n=8/group). An array of behavioral and cognitive tests were performed. All mice were sacrificed at 28 days after MCAO. Fluorescence-activated cell sorting (FACS) in combination with miRNA sequencing were used to profile the miRNA alteration. Results: Compared to ischemic reporter mice, ischemic Dicer-KO mice exhibited significantly increased infarct volumes and significantly impaired neurological outcomes and cognitive deficits post stroke as assayed by social recognition memory, novel object recognition, and Morris water-maze tests. Immunochemistry analysis revealed that ischemic Dicer-KO mice showed a significant reduction of the number of PLP-lineage NG2 + oligodendrocyte precursor cells (OPCs) and APC + myelinating oligodendrocytes compared to ischemic reporter mice. Moreover, Dicer-KO mice exhibited a robust reduction of myelinated axons measured by myelin binding protein (MBP) and Bielschowsky/Luxol Fast Blue assay. FACS analysis of PLP-lineage cells in peri-infarct regions showed that ablation of Dicer significantly decreased many miRNAs including the miR-200 family that are known to regulate oligodendrogenesis. Conclusion: Our data indicate that Dicer in PLP lineage oligodendrocytes is required for stroke-induced oligodendrogenesis and myelination, and that Dicer is involved in spontaneous functional recovery after stroke.

The LIN28B/TGF-β/TGFBI feedback loop promotes cell migration and tumour initiation potential in cholangiocarcinoma

Cholangiocarcinoma (CCA), a lethal malignancy of the biliary epithelium, is the second most common primary liver cancer. The poor prognosis of CCA is due to the high rate of tumour invasion and distant metastasis. We found that the RNA-binding protein LIN28B, a known regulator of microRNA biogenesis, stem cell maintenance, and oncogenesis, is expressed in a subpopulation of CCA patients. To further investigate the potential role of LIN28B in CCA pathogenesis, we studied the effect of LIN28B overexpression in the cholangiocyte cell line MMNK-1 and cholangiocarcinoma cell lines HuCCT-1 and KKU-214. Here, we show that enhanced LIN28B expression promoted cancer stem cell-like properties in CCA, including enhanced cell migration, epithelial-to-mesenchymal transition (EMT), increased cell proliferation and spheroid formation. Proteomic analysis revealed TGF-β-induced protein (TGFBI) as a novel LIN28B target gene, and further analysis showed upregulation of other components of the TGF-β signalling pathway, including TGF-β receptor type I (TGFBRI) expression and cytokine TGFB-I, II and III secretion. Importantly, the small molecule TGF-β inhibitor SB431542 negated the effects of LIN28B on both cell migration and clonogenic potential. Overexpression of TGFBI alone promoted cholangiocarcinoma cell migration and EMT changes, but not spheroid formation, suggesting that TGFBI partially contributes to LIN28B-mediated aggressive cell behaviour. These observations are consistent with a model in which TGF-β and LIN28B work together to form a positive feedback loop during cholangiocarcinoma metastasis and provide a therapeutic intervention opportunity.

Regulation of PKR activation and apoptosis during oxidative stress by TRBP phosphorylation.

Transactivation response element RNA-binding protein (TRBP or TARBP2) originally identified as a pro-viral cellular protein in human immunodeficiency virus (HIV) replication is also a regulator of microRNA biogenesis and cellular stress response. TRBP inhibits the catalytic activity of interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) during viral infections and cell stress thereby regulating stress-induced signaling pathways. During cellular stress, PKR is catalytically activated transiently by its protein activator PACT and TRBP inhibits PKR to bring about a timely cellular recovery. We have previously established that TRBP phosphorylated after oxidative stress binds to and inhibits PKR more efficiently promoting cell survival. In this study, we investigated if phosphorylation of TRBP enhances its interaction with PACT to bring about additional PKR inhibition. Our data establishes that phosphorylation of TRBP has no effect on PACT-TRBP interaction and TRBP's inhibitory actions on PKR are mediated exclusively by its enhanced interaction with PKR. Cells lacking TRBP are more sensitive to apoptosis in response to oxidative stress and show persistent PKR activation. These results establish that PKR inhibition by stress-induced TRBP phosphorylation occurs by its direct binding to PKR and is important for preventing apoptosis due to sustained PKR activation.

N6-methyladenosine demethylase ALKBH5 suppresses malignancy of esophageal cancer by regulating microRNA biogenesis and RAI1 expression.

N6-Methyladenosine (m6A) is the most prevalent epigenetic RNA modification and is vital in regulating malignancies. The roles of m6A modifiers on noncoding RNAs have not been fully investigated in esophageal cancer. By screening all m6A modifiers, ALKBH5 was the most potent member related to patient outcomes and suppressing esophageal cancer malignancy in cell and animal models. It demethylated pri-miR-194-2 and inhibited miR-194-2 biogenesis through an m6A/DGCR8-dependent manner. RAI1, previously considered as a circadian clock transcriptional regulator, was the main target of miR-194-2. It enhanced transcription of Hippo pathway upstream genes by binding to their 3'UTR and suppressed YAP/TAZ nuclear translocation. The ALKBH5/miR-194-2/RAI1 axis was also validated in clinical samples. In addition, the increased malignancy by low ALKBH5 was abolished by the YAP inhibitor verteporfin. Our findings uncover a critical role of ALKBH5 in miRNAs biogenesis and provide novel insight for developing treatment strategies in esophageal cancer.

Hypoxia-induced Nur77 activates PI3K/Akt signaling via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition.

Background: Colorectal cancer (CRC) and the associated metastatic lesions are reported to be hypoxic. Hypoxia is a common feature in the tumor microenvironment and a potent stimulant of CRC. We have identified a regulatory role of Nur77 on Akt activation to enhance β-catenin signaling essential for CRC progression under hypoxic conditions.Methods: The functional role of Nur77 in hypoxia-induced EMT was examined by scattering assays to monitor the morphologies of CRC cell lines under 1% O2. Sphere formation assays were performed to investigate whether Nur77 induced cancer stem cell-like properties in hypoxic CRC cells. The expression of various epithelial-to-mesenchymal transition (EMT) and stemness markers was analyzed by qPCR and Western blotting. Finally, Nur77 function and signaling in vivo was ascertained in subcutaneous tumor xenograft or liver metastasis model in nude mice using CRC cells stably transfected with appropriate constructs.Results: Herein, we show, for the first time, that Nur77 is a novel regulator of microRNA biogenesis that may underlie its significant tumor-promoting activities in CRC cells under hypoxia. Mechanistically, Nur77 interacted with the tumor suppressor protein p63, leading to the inhibition of p63-dependent transcription of Dicer, an important miRNA processor and subsequent decrease in the biogenesis of let-7i-5p which targeted the 3'UTR of p110α mRNA and regulated its stability. Knockdown of Nur77 or overexpression of let-7i-5p inhibited the tumor metastasis in vivo.Conclusion: Our data uncovered a novel mechanistic link connecting Nur77, Akt, and invasive properties of CRC in the hypoxic microenvironment.

Visualizing a protonated RNA state that modulates microRNA-21 maturation.

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. We show that the alternative excited conformation transiently sequesters the bulged adenine into a noncanonical protonated A+-G mismatch, conferring a substantial enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing a potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli.

Crosstalk of MicroRNAs and Oxidative Stress in the Pathogenesis of Cancer

Oxidative stress refers to an imbalance between reactive oxygen species (ROS) generation and body's capability to detoxify the reactive mediators or to fix the relating damage. MicroRNAs are considered to be important mediators that play essential roles in the regulation of diverse aspects of carcinogenesis. Growing studies have demonstrated that the ROS can regulate microRNA biogenesis and expression mainly through modulating biogenesis course, transcription factors, and epigenetic changes. On the other hand, microRNAs may in turn modulate the redox signaling pathways, altering their integrity, stability, and functionality, thus contributing to the pathogenesis of multiple diseases. Both ROS and microRNAs have been identified to be important regulators and potential therapeutic targets in cancers. However, the information about the interplay between oxidative stress and microRNA regulation is still limited. The present review is aimed at summarizing the current understanding of molecular crosstalk between microRNAs and the generation of ROS in the pathogenesis of cancer.

Complexity in regulating microRNA biogenesis in cancer.

The discovery of microRNA (miRNA) significantly extends our knowledge on gene regulation and noncoding gene functions. MiRNAs are important post-transcriptional regulators involve in a wide range of biological functions and diseases, including cancer. MiRNAs are produced by a unique biogenesis pathway involving the two-step sequential nuclear and cytoplasmic RNase-dependent processing at post-transcriptional level. However, a specific (set) of miRNA(s) is (are) synthesized under certain circumstance or developmental/pathological stage to fine-tune the gene expression profile. In this minireview, we will discuss the mechanism of miRNA biogenesis in cancer, mainly focusing on how Drosha and Dicer, two critical molecules controlling miRNA biogenesis, are modulated and which factor contributes to the specificity of selected miRNA maturation. Impact statement The canonical maturation pathway of miRNAs is highly conserved, indicating the crucial roles of these mini-regulators in most cellular processes. Dysregulation of specific miRNAs or imbalance of miRNA abundance has been observed in cancers. Accumulating evidence has shown that the interplay between miRNA processing factors and regulatory proteins previously known as key players in cancer malignancy regulates the biogenesis of miRNAs, expression of target genes, and eventually the alteration of cellular phenotypes. This minireview summarizes the current findings in the modulation of miRNA biogenesis in cancer to advance the understanding of how noncoding RNA contributes to cancer development and malignancy.

PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis.

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17–92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.

Engineering, delivery, and biological validation of artificial microRNA clusters for gene therapy applications.

The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.

Regulation of microRNA biogenesis and its crosstalk with other cellular pathways

MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. miRNAs are transcribed as precursor molecules, which are subsequently cleaved by the endoribonucleases Drosha and Dicer. Mature miRNAs are bound by a member of the Argonaute (AGO) protein family to form the RNA-induced silencing complex (RISC) in a process termed RISC loading. Advances in structural analyses of Drosha and Dicer complexes enabled elucidation of the mechanisms that drive these molecular machines. Transcription of miRNAs, their processing by Drosha and Dicer and RISC loading are key steps in miRNA biogenesis, and various additional factors facilitate, support or inhibit these processes. Recent work has revealed that regulatory factors not only coordinate individual miRNA processing steps but also connect miRNA biogenesis with other cellular processes. Protein phosphorylation, for example, links miRNA biogenesis to various signalling pathways, and such modifications are often associated with disease. Furthermore, not all miRNAs follow canonical processing routes, and many non-canonical miRNA biogenesis pathways have recently been characterized.

Targeting Pin1 by inhibitor API-1 regulates microRNA biogenesis and suppresses hepatocellular carcinoma development.

Experimental evidence suggests that Pin1 inhibition by API-1 up-regulates miRNA biogenesis by retaining active XPO5 conformation and suppresses HCC development, revealing the mechanism of Pin1-mediated miRNA biogenesis and unequivocally supporting API-1 as a drug candidate for HCC therapy, especially for Pin1-overexpressing, extracellular signal-regulated kinase-activated HCC. (Hepatology 2018).

ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

ADAR1 (adenosine deaminase acting on double-stranded RNA 1) is an RNA-editing enzyme that mediates adenosine-to-inosine RNA editing events, an important post-transcriptional modification mechanism that can alter the coding properties of mRNA or regulate microRNA biogenesis. ADAR1 also regulates the innate immune response. Here, we have demonstrated that ADAR1 expression increased in LPS-stimulated macrophages. Silencing ADAR1 by using small interfering RNA in macrophages resulted in the pronounced polarization of macrophages to M1, whereas ADAR1 overexpression promoted M2 polarization, which indicated that ADAR1 can inhibit macrophage hyperpolarization and prevent immune hyperactivity. The RNA-RNP immunoprecipitation binding assay demonstrated a direct interaction between ADAR1 and miR-21 precursor. Significant up-regulation in IL-10 and down-regulation in miR-21 were observed in ADAR1-overexpressing macrophages. We evaluated miR-21 target mRNAs and macrophage polarization signaling pathways and found that forkhead box protein O1 (Foxo1) was up-regulated in cells that overexpressed ADAR1. In a mouse allogeneic skin transplantation model, grafts in the ADAR1-overexpressed group survived longer and suffered less immune cell infiltration. In ADAR1-overexpressed recipients, splenic macrophages were significantly polarized to M2, and levels of sera IL-10 were markedly higher than those in the control group. In summary, ADAR1 modulates macrophage M2 polarization via the ADAR1-miR-21-Foxo1-IL-10 axis, thereby suppressing allogeneic graft rejection.-Li, J., Xie, J., Liu, S., Li, X., Zhang, D., Wang, X., Jiang, J., Hu, W., Zhang, Y., Jin, B., Zhuang, R., Yin, W. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

TSC2 regulates microRNA biogenesis via mTORC1 and GSK3β.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by germline inactivating mutations of TSC1 or TSC2. In TSC-associated tumors of the brain, heart, skin, kidney and lung, inactivation of both alleles of TSC1 or TSC2 leads to hyperactivation of the mTORC1 pathway. The TSC/mTORC1 pathway is a key regulator of cellular processes related to growth, proliferation and autophagy. We and others have previously found that mTORC1 regulates microRNA biogenesis, but the mechanisms are not fully understood. Microprocessor, a multi-protein complex including the nuclease Drosha, processes the primary miR transcript. Using a dual-luciferase reporter, we found that inhibition of mTORC1 or downregulation of Raptor decreased Microprocessor activity, while loss of TSC2 led to a striking increase (∼5-fold) in Microprocessor activity. To determine the global impact of TSC2 on microRNAs we quantitatively analyzed 752 microRNAs in Tsc2-expressing and Tsc2-deficient cells. Out of 259 microRNAs expressed in both cell lines, 137 were significantly upregulated and 24 were significantly downregulated in Tsc2-deficient cells, consistent with the increased Microprocessor activity. Microprocessor activity is known to be regulated in part by GSK3β. We found that total GSK3β levels were higher in Tsc2-deficient cells, and the increase in Microprocessor activity associated with Tsc2 loss was reversed by three different GSK3β inhibitors. Furthermore, mTOR inhibition increased the levels of phospho-GSK3β (S9), which negatively affects Microprocessor activity. Taken together these data reveal that TSC2 regulates microRNA biogenesis and Microprocessor activity via GSK3β.

Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

Transactivation response element RNA-binding protein (TRBP or TARBP2) initially identified to play an important role in human immunodeficiency virus (HIV) replication also has emerged as a regulator of microRNA biogenesis. In addition, TRBP functions in signaling pathways by negatively regulating the interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) during viral infections and cell stress. During cellular stress, PKR is activated and phosphorylates the α subunit of the eukaryotic translation factor eIF2, leading to the cessation of general protein synthesis. TRBP inhibits PKR activity by direct interaction as well as by binding to PKR’s two known activators, dsRNA and PACT, thus preventing their interaction with PKR. In this study, we demonstrate for the first time that TRBP is phosphorylated in response to oxidative stress and upon phosphorylation, inhibits PKR more efficiently promoting cell survival. These results establish that PKR regulation through stress-induced TRBP phosphorylation is an important mechanism ensuring cellular recovery and preventing apoptosis due to sustained PKR activation.

Genetic variation and RNA structure regulate microRNA biogenesis

MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast and gastric cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlight that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.

A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used aproteomics-based pull-down approach to map andcharacterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.