miRNA Processing Research Articles

The association between genetic variation rs2292832 and the processing efficiency of pre-mir-149 affects the risk of breast cancer.

microRNAs (miRNAs) play key roles in regulating cancer development, including breast cancer. Variation in miRNA genes can associate with the risk of cancer by alterations in the miRNA's processing and maturation. Therefore, human blood samples and breast cancer cell line (MCF7) were analyzed to study any possible association between the genetic variant (rs2292832) in the miR-149 precursor and breast cancer susceptibility. To study the role of rs2292832 polymorphism in breast cancer, the miR-149 gene variant was genotyped using PCR-RFLP. For evaluating the effect of SNP on function and expression levels of mature miR-149, we inserted pre-miR-149 and flanking region with CC or TT genotype into a pEGFPN1 expression vector, and qPCR was accomplished. Cell survival, proliferation, and migration properties investigated by MTT and wound healing assay. Statistical analysis was carried out for data analysis. T allele in variant rs2292832 is associated with an increased risk of breast cancer. Such association was also obtained in co-dominant (OR = 2.5) and dominant (OR = 2.016) models. The variant allele led to reduced production of mature miR-149 and resulted in increased cell proliferation and migration of MCF7 cells. These findings suggest that miR-149 suppresses tumor cell proliferation, and the pre-mir-149 polymorphism affects the processing of miR-149, causing an alteration in the abundance of the miRNA mature form, which can regulate tumor progression and metastasis.

Abstract 11998: ADAR1 Mediates Macrophage Activation by Its Non-Editing Activity-Mediated Pri-miRNA Processing in Abdominal Aortic Aneurysm

Introduction: Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation. Methods: Aortic transplantation was conducted to determine the importance of non-vascular ADAR1 in AAA development. Angiotensin II (Ang II) infusion of ApoE–/– mouse combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation. Primary cultured bone-marrow derived and peritoneal macrophages were used to study how ADAR1 regulates macrophage activation. Moreover, a novel humanized AAA model was established by transplanting human artery to immunodeficient mouse aorta followed by immunity reconstitution with ADAR1-deficient human monocytes to test the role of human macrophages in aneurysm formation in human arteries. Results: Allograft transplantation of wild type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that non-vascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB signaling, and enhanced the expression of many anti-inflammatory miRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 interaction, and consequently inhibited pri- to pre-miRNA processing of miRNAs targeting IKKβ, resulting in an increased IKKβ expression and enhanced NF-κB signaling. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Conclusions: Macrophage ADAR1 promotes AAA formation in both mouse and human arteries through a novel mechanism, i.e., Drosha protein degradation, which inhibits the processing of miRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation.

Association analysis of miRNA-146a and miRNA-499 polymorphisms with rheumatoid arthritis: a case-control and trio-family study.

Single nucleotide polymorphism is known to alter the expression and processing of miRNAs leading to a variety of diseases including rheumatoid arthritis (RA). However, disagreement is present up to date regarding the association of miRNA-146a and miRNA-499 polymorphisms with RA. The goal of this study was to assess the association of polymorphisms at miRNA-146a and miRNA-499 with the pathogenesis of RA in patients originating from Pakistan. Initially, eleven hundred subjects (1100) comprises of 550 RA patients and 550 healthy controls were investigated in the case-control analysis. Spectrophotometric measurement of lipids and C-reactive protein was used, whereas interleukin-1 receptor associated kinase-1 and TNF-receptor associated factor-6 values were quantified by an enzyme-linked immunosorbent assay. Secondly, heritability of susceptible alleles was tested from 70 trio-families. The miRNA-146a rs2910164 and miRNA-499 rs3746444 polymorphisms were genotyped using the polymerase chain reaction followed by restriction digestion. A Significant association of miRNA-146a and miRNA-499 genotypes was observed with RA patients (P < 0.05, respectively). The miRNA-146a rs2910164 G (OR = 1.4, P < 0.05) and miRNA-499 rs3746444 C (OR = 1.6, P < 0.0001) allele was significantly associated with RA in comparison with controls, respectively. Besides, the transmission analysis revealed a significant (P < 0.05) inheritance of rs2910164 G and rs3746444 C allele from parents to affected offspring. The current research concludes that miRNA-146a (rs2910164; C > G) and miRNA-499 (rs3746444; T > C) polymorphisms are linked to RA in the population studied. Furthermore, it was demonstrated for the first time in our high-risk cohort that the rs2910164 G and rs3746444 C allele was strongly related to familial RA.

DAZL regulates proliferation of human primordial germ cells by direct binding to precursor miRNAs and enhances DICER processing activity.

Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA-binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cell-specific RBP, in miRNA biogenesis and cell proliferation. First, DAZL co-localized with miRNA let-7a in human PGCs and up-regulated the levels of &gt;100 mature miRNAs, including eight out of nine let-7 family, miR21, miR22, miR125, miR10 and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs up-regulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline tumor cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing.

HnRNP A1 in RNA metabolism regulation and as a potential therapeutic target.

Abnormal RNA metabolism, regulated by various RNA binding proteins, can have functional consequences for multiple diseases. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an important RNA binding protein, that regulates various RNA metabolic processes, including transcription, alternative splicing of pre-mRNA, translation, miRNA processing and mRNA stability. As a potent splicing factor, hnRNP A1 can regulate multiple splicing events, including itself, collaborating with other cooperative or antagonistical splicing factors by binding to splicing sites and regulatory elements in exons or introns. hnRNP A1 can modulate gene transcription by directly interacting with promoters or indirectly impacting Pol II activities. Moreover, by interacting with the internal ribosome entry site (IRES) or 3'-UTR of mRNAs, hnRNP A1 can affect mRNA translation. hnRNP A1 can alter the stability of mRNAs by binding to specific locations of 3'-UTR, miRNAs biogenesis and Nonsense-mediated mRNA decay (NMD) pathway. In this review, we conclude the selective sites where hnRNP A1 binds to RNA and DNA, and the co-regulatory factors that interact with hnRNP A1. Given the dysregulation of hnRNP A1 in diverse diseases, especially in cancers and neurodegeneration diseases, targeting hnRNP A1 for therapeutic treatment is extremely promising. Therefore, this review also provides the small-molecule drugs, biomedicines and novel strategies targeting hnRNP A1 for therapeutic purposes.

Ozone and nitrogen dioxide regulate similar gene expression responses in Arabidopsis but natural variation in the extent of cell death is likely controlled by different genetic loci.

High doses of ozone (O3) and nitrogen dioxide (NO2) cause damage and cell death in plants. These two gases are among the most harmful air pollutants for ecosystems and therefore it is important to understand how plant resistance or sensitivity to these gases work at the molecular level and its genetic control. We compared transcriptome data from O3 and NO2 fumigations to other cell death related treatments, as well as individual marker gene transcript level in different Arabidopsis thaliana accessions. Our analysis revealed that O3 and NO2 trigger very similar gene expression responses that include genes involved in pathogen resistance, cell death and ethylene signaling. However, we also identified exceptions, for example RBOHF encoding a reactive oxygen species producing RESPIRATORY BURST OXIDASE PROTEIN F. This gene had increased transcript levels by O3 but decreased transcript levels by NO2, showing that plants can identify each of the gases separately and activate distinct signaling pathways. To understand the genetics, we conducted a genome wide association study (GWAS) on O3 and NO2 tolerance of natural Arabidopsis accessions. Sensitivity to both gases seem to be controlled by several independent small effect loci and we did not find an overlap in the significantly associated regions. Further characterization of the GWAS candidate loci identified new regulators of O3 and NO2 induced cell death including ABH1, a protein that functions in abscisic acid signaling, mRNA splicing and miRNA processing. The GWAS results will facilitate further characterization of the control of programmed cell death and differences between oxidative and nitrosative stress in plants.

Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma.

Simple SummaryThe gene of the human tumor suppressive microRNA-142 (miR-142) carries mutations in about 20% of cases of diffuse large B-cell lymphoma (DLBCL). Because microRNAs post-transcriptionally regulate the protein expression of their cognate messenger RNA (mRNAs) targets, we determined the effect of miR-142 knockout on protein expression in two cell lines derived from DLBCL. We found a significant up-regulation of 52 proteins but also a down-regulation of 41 proteins upon miR-142 deletion. Knockout of a miRNA may be used to identify novel targets, and seed-sequence mutants of a miRNA unable to bind to their targets can be used to confirm potential novel targets. With this approach, we identify AKT1S1, CCNB1, LIMA1 and TFRC as novel targets of miR-142. As miR-142 is highly present in the miRNA processing RISC complexes, the deletion of this miRNA might result in its replacement by other miRNAs, thus introducing an additional layer of complexity regarding gene regulation.Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation.

Identification of a miRSNP Regulatory Axis in Abdominal Aortic Aneurysm by a Network and Pathway-Based Integrative Analysis.

Abdominal aortic aneurysm (AAA) refers to local abnormal expansion of the abdominal aorta and mostly occurs in elderly men. MicroRNA (miRNA) is single-stranded RNA consisting of 18–25 nucleotides. It plays a key role in posttranscriptional gene expression and in the regulation of human functions and disease development. miRNA exerts its function mainly through the binding of complementary base pairs to the 3′ regulatory region of mRNA transcripts. Therefore, miRNA-related single-nucleotide polymorphisms (miRSNPs) can affect miRNA expression and processing kinetics. miRSNPs can be classified based on their location: miRSNPs within miRNA-producing genes and miRSNPs within miRNA target genes. Increasing evidence indicates that miRSNPs play an important role in the pathogenic kinetics of cardiovascular diseases. The aim of this study was to identify potential miRNAs and integrate them into a miRSNP-based disease-related pathway network, the results of which are of great significance to the interpretation of the potential mechanisms and functions of miRSNPs in the pathogenesis of diseases.

Miniature Inverted-repeat Transposable Elements Drive Rapid MicroRNA Diversification in Angiosperms.

MicroRNAs (miRNAs) are fast evolving endogenous small RNAs that regulate organism function and behavior in both animals and plants. Although models for de novo miRNA biogenesis have been proposed, the genomic mechanisms driving swift diversification of the miRNA repertoires in plants remain elusive. Here, by comprehensively analyzing 21 phylogenetically representative plant species, ranging from green algae to angiosperms, we systematically identified de novo miRNA events associated with 8,649 miRNA loci. We found that 399 (4.6%), 466 (5.4%), and 1,402 (16.2%) miRNAs were derived from inverted gene duplication events, long terminal repeats of retrotransposons, and miniature inverted-repeat transposable elements (MITEs), respectively. Among the miRNAs of these origins, MITEs, especially those belonging to the Mutator, Tc1/Mariner, and PIF/Harbinger superfamilies, were the predominant genomic source for de novo miRNAs in the 15 examined angiosperms but not in the six non-angiosperms. Our data further illustrated a transposition-transcription process by which MITEs are converted into new miRNAs (termed MITE-miRNAs) whereby properly sized MITEs are transcribed and therefore become potential substrates for the miRNA processing machinery by transposing into introns of active genes. By analyzing the 58,038 putative target genes for the 8,095 miRNAs, we found that the target genes of MITE-miRNAs were preferentially associated with response to environmental stimuli such as temperature, suggesting that MITE-miRNAs are pertinent to plant adaptation. Collectively, these findings demonstrate that molecular conversion of MITEs is a genomic mechanism leading to rapid and continuous changes to the miRNA repertoires in angiosperm.

Abstract A18: GRK2, an inhibitor of MALT1-dependent oncogenic signaling, is downregulated by microRNA in ABC-DLBCL

Abstract Introduction and Background: The “CBM” complex is composed of the scaffolding protein CARMA1, the adaptor protein BCL10, and the effector protein MALT1. This complex performs multiple pivotal functions as a mediator of antigen receptor-dependent induction of the NF-κB transcription factor and subsequent lymphocyte activation. The key downstream effector of the CBM complex, MALT1, regulates downstream signaling via both scaffolding and proteolytic functions. Inappropriate activation of MALT1, which can result from somatic gain-of-function mutation in upstream regulators of MALT1 or chromosomal translocation involving the MALT1 gene, underlies the pathogenesis of a variety of lymphoid malignancies including activated B cell type-diffuse large B cell lymphoma (ABC-DLBCL) and mucosa associated lymphoid tissue (MALT) lymphoma. We recently discovered that G-protein-coupled receptor kinase 2 (GRK2) binds to MALT1 and inhibits MALT1 scaffold and proteolytic activities. Further, we found that knockdown of GRK2 in ABC-DLBCL enhances tumor growth in vivo, suggesting that GRK2 may act as a tumor suppressor in MALT1-dependent lymphomas. Interestingly, we found that GRK2 mRNA levels are markedly lower in a subset of DLBCL cases in comparison to normal B-cell controls and that lower GRK2 level is associated with reduced survival in ABC-DLBCL patients. We thus sought to investigate how GRK2 expression is regulated in ABC-DLBCL. Methods and Results: We first investigated whether mutations/deletions in the ADRBK1 gene (GRK2) account for the lower levels of GRK2 expression in a subset of DLBCL cases. Only one mutation among 6 published DLBCL sequencing datasets (334 cases total) was identified. We next considered that other mechanisms, such as regulation by microRNA (miRNA), could play a role in downregulating GRK2. Using DICER-deficient HEK 293T cells, we demonstrated that GRK2 protein level significantly increases when miRNA processing is impaired. We then used mirDIP, a microRNA Data Integration Portal, to screen for candidate miRNAs predicted to target the 3’UTR of GRK2. We identified 4 top candidate hits, 2 of which were also identified in bioinformatic analysis as miRNA whose level inversely correlates with GRK2 mRNA level in ABC-DLBCL patient tumor specimens. Using a GRK2 3’ UTR reporter system, we confirmed direct targeting of GRK2 by these miRNAs. Next, we generated stable ABC-DLBCL cell lines (TMD8, OCI-Ly3) overexpressing candidate miRNAs. Our results demonstrate that candidate miRNA overexpression results in reduced GRK2 expression and increased MALT1 scaffolding and proteolytic activities, NF-κB transcriptional activity and ABC-DLBCL cell proliferation. Conclusions: Together, our data suggest that miRNAs down-regulate expression of GRK2 in ABC-DLBCL which in turn enhances MALT1 scaffolding and proteolytic activities, leading to increased tumor cell proliferation. Future studies are aimed at identifying potential miRNA inhibitors which enhance GRK2 expression and thereby suppress MALT1 pro-tumorigenic activity in ABC-DLBCL. Citation Format: Jing Cheng, Mei Smyers, Matt Trotta, Neil M Carleton, Lisa M Maurer, Peter C Lucas, Linda M McAllister-Lucas. GRK2, an inhibitor of MALT1-dependent oncogenic signaling, is downregulated by microRNA in ABC-DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A18.

Methyltransferase-like 3 (METTL3) mediated N6-methyladenosine (m6A) modifications facilitate mir-25-3p maturation to promote gastrointestinal stromal tumors (GISTs) progression.

Methyltransferase-like 3 (METTL3) is an RNA N6-methyladenosine (m6A) methyltransferase, which plays a critical role in micorRNA (miRNAs) processing and maturation, but it is still unclear whether METTL3 regulated miRNAs participates in the regulation of cancer aggressiveness in gastrointestinal stromal tumors (GISTs). This study was designed to investigate this issue, and uncover the potential underlying mechanisms. the expression of METTL3 in GISTs tissues and cell lines were determined by RT-qPCR and Western blot. Cell proliferation and migration were assessed by colony formation, CCK-8 and Transwell. The mRNA expression of all proteins was detected by RT-qPCR, and tumor xenograft study was applied to confirm the effect of METTL3 on GISTs development in vivo. In our study, we showed that METTL3 was significantly upregulated in GISTs tissues and cell lines. Functional experiments demonstrated that overexpression of METTL3 promoted GISTs cell malignant biological behavior and tumor growth in vitro and in vivo, and conversely, silencing of METTL3 had opposite effects and suppressed GISTs progression. Further mechanistical experiments verified that METTL3 promoted the maturation of miR-25-3p in an m6A-dependent manner. Similar to METTL3, miR-25-3p was also validated as an oncogene to promote cancer development in GISTs. Finally, our rescuing experiments hinted that silencing of miR-25-3p abrogated the tumor-initiating effects of METTL3 overexpression on GISTs. Collectively, those results indicated that METTL3 played an oncogenic role in GISTs through positively modulating the miR-25-3p in an m6A-dependent manner, and we firstly discussed how the METTL3/m6A/miR-25-3p axis affected GISTs development.

Identification of Pri-miRNA Stem-Loop Interacting Proteins in Plants Using a Modified Version of the Csy4 CRISPR Endonuclease.

Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (miRNAs) is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long primary-microRNAs (pri-miRNAs) with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs.

Dicer-mediated miR-200b expression contributes to cell migratory/invasive abilities and cancer stem cells properties of breast cancer cells.

Distant metastasis is the leading cause of death in patients with breast cancer. Despite considerable treatment advances, the clinical outcomes of patients with metastatic breast cancer remain poor. CSCs can self-renew, enhancing cancer progression and metastasis. Dicer, a microRNA (miRNA) processing–related enzyme, is required for miRNA maturation. Imbalanced Dicer expression may be pivotal in cancer progression. However, whether and how Dicer affects the stemness of metastatic breast cancer cells remains unclear. Here, we hypothesized that Dicer regulates the migration, invasion, and stemness of breast cancer cells. We established highly invasive cell lines (MCF-7/I-3 and MDA-MB-231/I-3) and observed that Dicer expression was conspicuously lower in the highly invasive cells than in the parental cells. The silencing of Dicer significantly enhanced the cell migratory/invasive abilities and CSCs properties of the breast cancer cells. Conversely, the overexpression of Dicer in the highly invasive cells reduced their migration, invasion, and CSCs properties. Our bioinformatics analyses demonstrated that low Dicer levels were correlated with increased breast cancer risk. Suppression of Dicer inhibited miR-200b expression, whereas miR-200b suppression recovered Dicer knockdown–induced migration, invasion, and cancer stem cells (CSCs) properties of the breast cancer cells. Thus, our findings reveal that Dicer is a crucial regulator of the migration, invasion, and CSCs properties of breast cancer cells and is significantly associated with poor survival in patients with breast cancer.

Spotting the Targets of the Apospory Controller TGS1 in Paspalum notatum.

Sexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis.

Single-molecule FRET uncovers hidden conformations and dynamics of human Argonaute 2

Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide’s 3’-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle.

PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing.

DICER1 syndrome is a cancer pre-disposition disorder caused by mutations that disrupt the function of DICER1 in miRNA processing. Studying the molecular, cellular and oncogenic effects of these mutations can reveal novel mechanisms that control cell homeostasis and tumor biology. Here, we conduct the first analysis of pathogenic DICER1 syndrome allele from the DICER1 3′UTR. We find that the DICER1 syndrome allele, rs1252940486, abolishes interaction with the PUMILIO RNA binding protein with the DICER1 3′UTR, resulting in the degradation of the DICER1 mRNA by AUF1. This single mutational event leads to diminished DICER1 mRNA and protein levels, and widespread reprogramming of miRNA networks. The in-depth characterization of the rs1252940486 DICER1 allele, reveals important post-transcriptional regulatory events that control DICER1 levels.

Molecular Dissection of a Conserved Cluster of miRNAs Identifies Critical Structural Determinants That Mediate Differential Processing.

Differential processing is a hallmark of clustered microRNAs (miRNAs) and the role of position and order of miRNAs in a cluster together with the contribution of stem-base and terminal loops has not been explored extensively within the context of a polycistronic transcript. To elucidate the structural attributes of a polycistronic transcript that contribute towards the differences in efficiencies of processing of the co-transcribed miRNAs, we constructed a series of chimeric variants of Drosophila let-7-Complex that encodes three evolutionary conserved and differentially expressed miRNAs (miR-100, let-7 and miR-125) and examined the expression and biological activity of the encoded miRNAs. The kinetic effects of Drosha and Dicer processing on the chimeric precursors were examined by in vitro processing assays. Our results highlight the importance of stem-base and terminal loop sequences in differential expression of polycistronic miRNAs and provide evidence that processing of a particular miRNA in a polycistronic transcript is in part determined by the kinetics of processing of adjacent miRNAs in the same cluster. Overall, this analysis provides specific guidelines for achieving differential expression of a particular miRNA in a cluster by structurally induced changes in primary miRNA (pri-miRNA) sequences.

The long non-coding RNA MEG8 induces an endothelial barrier through regulation of microRNA-370 and -494 processing

ABSTRACTThe 14q32 locus is an imprinted region in the human genome which contains multiple non-coding RNAs. We investigated the role of the long non-coding RNA maternally expressed gene 8 (MEG8) in endothelial function and its underlying mechanism. A 5-fold increase in MEG8 was observed with increased passage number in human umbilical vein endothelial cells (HUVECs), suggesting MEG8 is induced during aging. MEG8 knockdown resulted in a 1.8-fold increase in senescence, suggesting MEG8 might be protective during aging. The endothelial barrier was also impaired after MEG8 silencing. MEG8 knockdown resulted in reduced expression of microRNA (miRNA)-370 and -494 but not -127, -487b and -410. Overexpression of miRNA-370 or -494 partially rescued the MEG8-silencing-induced barrier loss. Mechanistically, MEG8 regulates expression of miRNA-370 and -494 at the mature miRNA level through interaction with the RNA-binding proteins cold-inducible RNA-binding protein (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multi-enzyme complex subunit β (HADHB). Mature miRNA-370 and miRNA-494 were found to interact with CIRBP, whereas precursor miRNA-370 and miRNA-494 were found to interact with HADHB. Individual CIRBP and HADHB silencing resulted in downregulation of miRNA-370 and induction of miRNA-494. These results suggest MEG8 interacts with CIRBP and HADHB and contributes to miRNA processing at the post-transcriptional level.

A Novel miRNA Located in the HER2 Gene Shows an Inhibitory Effect on Wnt Signaling and Cell Cycle Progression.

Human epidermal growth factor receptor 2 (HER2) is involved in the development of the majority of cancers. Therefore, it can be a potential target for cancer therapy. It was hypothesized that some of the broad effects of HER2 could be mediated by miRNAs that are probably embedded inside this gene. Here, we predicted and then empirically substantiated the processing and expression of a novel miRNA named HER2-miR1, located in the HER2 gene; transfection of a DNA fragment corresponding to HER2-miR1 precursor sequence (preHER2-miR1) resulted in ~4000-fold elevation of HER2-miR1 mature form in HEK293t cells. Also, the detection of HER2-miR1 in 5637, NT2, and HeLa cell lines confirmed its endogenous production. Following the HER2-miR1 overexpression, TOP/FOP flash assay and RT-qPCR results showed that Wnt signaling pathway was downregulated. Consistently, flow cytometry results revealed that overexpression of HER2-miR1 in Wnt+ cell lines (SW480 and HCT116) was ended in G1 arrest, unlike in Wnt− cells (HEK293t). Taking everything into account, our results report the discovery of a novel miRNA that is located within the HER2 gene sequence and has a repressive impact on the Wnt signaling pathway.

Post-transcriptional regulation of polycistronic microRNAs.

An important proportion of microRNA (miRNA) genes tend to lie close to each other within animal genomes. Such genomic organization is generally referred to as miRNA clusters. Even though many miRNA clusters have been greatly studied, most attention has been usually focused on functional impacts of clustered miRNA co-expression. However, there is also another compelling aspect about these miRNA clusters, their polycistronic nature. Being transcribed on a single RNA precursor, polycistronic miRNAs benefit from common transcriptional regulation allowing their coordinated expression. And yet, numerous reports have revealed striking discrepancies in the accumulation of mature miRNAs produced from the same cluster. Indeed, the larger polycistronic transcripts can act as platforms providing unforeseen post-transcriptional regulatory mechanisms controlling individual miRNA processing, thus leading to differential miRNA expression, and sometimes even challenging the general assumption that polycistronic miRNAs are co-expressed. In this review, we aim to address the current knowledge about how miRNA polycistrons are post-transcriptionally regulated. In particular, we will focus on the mechanisms occurring at the level of the primary transcript, which are highly relevant for individual miRNA processing and as such have a direct repercussion on miRNA function within the cell. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.