scholarly journals Viral miRNA adaptor differentially recruits miRNAs to target mRNAs through alternative base-pairing

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Carlos Gorbea ◽  
Tim Mosbruger ◽  
David A Nix ◽  
Demián Cazalla
Keyword(s):  
Seed Region ◽  
Base Pairing ◽  
Recognition Mechanism ◽  
Target Mrnas ◽  
Non Coding Rna ◽  
Infected Cells ◽  
Nucleotide Resolution ◽  
Rna Interaction ◽  
General Method

HSUR2 is a viral non-coding RNA (ncRNA) that functions as a microRNA (miRNA) adaptor. HSUR2 inhibits apoptosis in infected cells by recruiting host miRNAs miR-142–3p and miR-16 to mRNAs encoding apoptotic factors. HSUR2’s target recognition mechanism is not understood. It is also unknown why HSUR2 utilizes miR-16 to downregulate only a subset of transcripts. We developed a general method for individual-nucleotide resolution RNA-RNA interaction identification by crosslinking and capture (iRICC) to identify sequences mediating interactions between HSUR2 and target mRNAs in vivo. Mutational analyses confirmed identified HSUR2-mRNA interactions and validated iRICC as a method that confidently determines sequences mediating RNA-RNA interactions in vivo. We show that HSUR2 does not display a ‘seed’ region to base-pair with most target mRNAs, but instead uses different regions to interact with different transcripts. We further demonstrate that this versatile mode of interaction via variable base-pairing provides HSUR2 with a mechanism for differential miRNA recruitment.

scholarly journals Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA

2021 ◽  
Author(s):  
Ye Duan ◽  
Isana Veksler-Lublinsky ◽  
Victor Ambros
Keyword(s):  
Systematic Investigation ◽  
Seed Region ◽  
Seed Sequence ◽  
Base Pairing ◽  
Non Coding Rna ◽  
Evolutionarily Conserved ◽  
Microrna Function

MicroRNAs are endogenous regulatory non-coding RNA that exist in all multi-cellular organisms. Base-pairing of the seed region (g2-g8) is essential for microRNA targeting, however the in vivo functions of 3' non-seed region (g9-g22) are less well understood. Here we report the first systematic investigation of the in vivo roles of 3' non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We found that the 3' non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets in vivo, including evolutionarily conserved lin-41, daf-12 and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, we find that 3' non-seed pairing of let-7a can be functionally required even with sites that permit perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3' non-seed base-pairing can critically influence microRNA function in vivo.

scholarly journals A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency

2014 ◽  
Vol 42 (15) ◽  
pp. 10050-10060 ◽  
Author(s):  
Hideaki Kume ◽  
Kimihiro Hino ◽  
Josephine Galipon ◽  
Kumiko Ui-Tei
Keyword(s):  
Gene Expression Regulation ◽  
Seed Region ◽  
Rna Sequences ◽  
Protein Coding ◽  
Adenosine Deaminases ◽  
Target Mrnas ◽  
Non Coding Rna ◽  
The Difference ◽  
Mirna Seed

Abstract Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding machinery. Genome-wide studies have revealed that the majority of editing events occur in non-coding RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs that regulate the expression of target mRNAs with complementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex globally reassigns their target mRNAs in vivo, and reveal that miRNA containing inosine in the seed region exhibits a different degree of silencing efficiency compared to the corresponding miRNA with guanosine at the same position. The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

scholarly journals Structure of anEscherichia coliHfq:RNA complex at 0.97 Å resolution

2014 ◽  
Vol 70 (11) ◽  
pp. 1492-1497 ◽  
Author(s):  
Eike C. Schulz ◽  
Orsolya Barabas
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Binding Site ◽  
Small Rnas ◽  
Target Genes ◽  
Rna Binding ◽  
Base Pairing ◽  
Rna Chaperone ◽  
Target Mrnas ◽  
Rna Interaction

In bacteria, small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA, thereby modulating its translation. A central player in this process is the RNA chaperone Hfq, which facilitates the annealing of sRNAs with their target mRNAs. Hfq has two RNA-binding surfaces that recognize A-rich and U-rich sequences, and is believed to bind an sRNA–mRNA pair simultaneously. However, how Hfq promotes annealing remains unclear. Here, the crystal structure ofEscherichia coliHfq is presented in complex with U6-RNA bound to its proximal binding site at 0.97 Å resolution, revealing the Hfq–RNA interaction in exceptional detail.

KRAB Zinc Finger protein Znf684 interacts with Nxf1 to regulate mRNA export

2021 ◽  
Author(s):  
Alexandra Nitoiu ◽  
Syed Nabeel-Shah ◽  
Shaghayegh Farhangmehr ◽  
Shuye Pu ◽  
Ulrich Braunschweig ◽  
...  
Keyword(s):  
Zinc Finger ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Affinity Purification ◽  
Mrna Export ◽  
Target Mrnas ◽  
Specific Mrnas ◽  
Nucleotide Resolution

AbstractCys2His2 (C2H2) type zinc finger (ZnF) proteins constitute a large class of proteins that are generally considered to be DNA-binding transcription factors. Using affinity purification followed by mass spectrometry, as well as reciprocal co-immunoprecipitation experiments, we determined that the C2H2-ZnF protein Znf684 interacts physically with several proteins involved in mRNA export, including Nxf1 and Alyref. We utilized individual nucleotide resolution cross-linking immunoprecipitation followed by high throughput sequencing (iCLIP-seq) experiments to show that Znf684 binds directly to specific mRNAs in vivo and has an RNA-binding profile similar to those of Nxf1 and Alyref, suggesting a role in mRNA export regulation. Immunofluorescence microscopy (IF) experiments revealed that Znf684 localizes to both the nucleus and cytoplasm. Using cellular fractionation experiments, we demonstrate that overexpression of Znf684 negatively impacts the export of SMAD3 and other target mRNAs. Taken together, our results suggest that Znf684 regulates the export of a subset of transcripts through physical interactions with Nxf1 and specific target mRNAs.

scholarly journals Insights into the secondary structural ensembles of the full SARS-CoV-2 RNA genome in infected cells

2021 ◽  
Author(s):  
Silvi Rouskin ◽  
Tammy Lan ◽  
Matthew Allan ◽  
Lauren Malsick ◽  
Stuti Khandwala ◽  
...  
Keyword(s):  
Rna Folding ◽  
Dimethyl Sulfate ◽  
Ribosome Profiling ◽  
Regulatory Sequences ◽  
Rna Structures ◽  
Infected Cells ◽  
Rna Genome ◽  
Nucleotide Resolution

Abstract SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on native RNA structures in SARS-CoV-2 and most putative regulatory sequences are functionally uncharacterized. Here we report secondary structure ensembles of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and the algorithm ‘detection of RNA folding ensembles using expectation–maximization’ clustering (DREEM). Our results reveal previously undescribed alternative RNA conformations across the genome, including structures of the frameshift stimulating element (FSE), a major drug target, that are drastically different from prevailing in vitro population average models. Importantly, we find that this structural ensemble promotes frameshifting rates (~40%) similar to in vivo ribosome profiling studies and much higher than the canonical minimal FSE (~20%). Overall, our result highlight the value of studying RNA folding in its native, dynamic and cellular context. The genomic structures detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

scholarly journals Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose

2018 ◽  
Author(s):  
Kristen M. Bartoli ◽  
Cassandra Schaening ◽  
Thomas M. Carlile ◽  
Wendy V. Gilbert
Keyword(s):  
List Type ◽  
Single Nucleotide ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Non Coding Rna ◽  
Genome Wide ◽  
Non Coding Rnas ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution ◽  
Specific Mrna

SUMMARYNon-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2′-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2′-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2′-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation.HIGHLIGHTSMeTH-seq identifies 2′-O-methylribose genome-wide at single-nucleotide resolutionFive conserved methyltransferases interact with yeast mRNASpb1 is a major mRNA 2′-O-methyltransferase, and targets most ribosomal protein mRNAsSPB1 expression is required to maintain normal levels of Spb1 target mRNAs

scholarly journals LncRNA PVT1 promotes cervical cancer progression by sponging miR-503 to upregulate ARL2 expression

2021 ◽  
Vol 16 (1) ◽  
pp. 1-13
Author(s):  
Weiwei Liu ◽  
Dongmei Yao ◽  
Bo Huang
Keyword(s):  
Cervical Cancer ◽  
Cancer Progression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Nude Mouse Model ◽  
Western Blot Assay ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract Cervical cancer (CC) is a huge threat to the health of women worldwide. Long non-coding RNA plasmacytoma variant translocation 1 gene (PVT1) was proved to be associated with the development of diverse human cancers, including CC. Nevertheless, the exact mechanism of PVT1 in CC progression remains unclear. Levels of PVT1, microRNA-503 (miR-503), and ADP ribosylation factor-like protein 2 (ARL2) were measured by quantitative reverse transcription-polymerase chain reaction or western blot assay. 3-(4,5)-Dimethylthiazole-2-y1)-2,5-biphenyl tetrazolium bromide (MTT) and flow cytometry were used to examine cell viability and apoptosis, respectively. For migration and invasion detection, transwell assay was performed. The interaction between miR-503 and PVT1 or ARL2 was shown by dual luciferase reporter assay. A nude mouse model was constructed to clarify the role of PVT1 in vivo. PVT1 and ARL2 expressions were increased, whereas miR-503 expression was decreased in CC tissues and cells. PVT1 was a sponge of miR-503, and miR-503 targeted ARL2. PVT1 knockdown suppressed proliferation, migration, and invasion of CC cells, which could be largely reverted by miR-503 inhibitor. In addition, upregulated ARL2 could attenuate si-PVT1-mediated anti-proliferation and anti-metastasis effects on CC cells. Silenced PVT1 also inhibited CC tumor growth in vivo. PVT1 knockdown exerted tumor suppressor role in CC progression via the miR-503/ARL2 axis, at least in part.

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