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Lanqing Ying, Hongkun Zhu, Shinichiro Shoji, Kurt Fredrick
Aminoglycosides containing a 2-deoxystreptamine core (AGs) represent a large family of antibiotics that target the ribosome. These compounds promote miscoding, inhibit translocation, and inhibit ribosome recycling. AG binding to helix h44 of the small subunit induces rearrangement of A-site nucleotides (nt) A1492 and A1493, which promotes a key open-to-closed conformational change of the subunit and thereby increases miscoding. Mechanisms by which AGs inhibit translocation and recycling remain less clear. Structural studies have revealed a secondary AG binding site in H69 of the large subunit, and it has been proposed that interaction at this site is crucial for inhibition of translocation and recycling...
November 9, 2018: RNA
Brian Yee, Gabriel Pratt, Brenton Graveley, Eric Van Nostrand, Gene Yeo
Alternative splicing of pre-messenger RNA transcripts enables the generation of multiple protein isoforms from the same gene locus, providing a major source of protein diversity in mammalian genomes. RNA binding proteins (RBPs) bind to RNA to control splice site choice and define which exons are included in the resulting mature RNA transcript. However, depending on where the RBPs bind relative to splice sites, they can activate or repress splice site usage. To explore this position-specific regulation, in vivo binding sites identified by methods such as cross-linking immunoprecipitation (CLIP) are integrated with alternative splicing events identified by RNA-seq or microarray...
November 9, 2018: RNA
Filip Leonarski, Luigi D'Ascenzo, Pascal Auffinger
Precise knowledge of Mg2+ inner-sphere binding site characteristics is vital for understanding the structure and function of nucleic acid systems. Unfortunately, the PDB, that represents the main source of Mg2+ binding sites, contains a significant number of ion assignment issues that significantly blur our understanding of the functions of these ions. Here, following a preceding study devoted to Mg2+ binding to nucleobase nitrogens, we surveyed PDB nucleic acid crystallographic structures with resolutions < 2...
November 8, 2018: RNA
Nicole Erlenbach, Christian Grünewald, Bisera Krstic, Alexander Heckel, Thomas F Prisner
PELDOR (pulse electron-electron double resonance) is an established method to study intramolecular distances and can give evidence for conformational changes and flexibilities. However, it can also be used to study intermolecular interactions as for example oligerimization. Here, we used PELDOR to study the 'end-to-end' stacking of small double stranded (ds)RNAs. For this study, the dsRNA molecules were only singly labelled with the spin label TPA to avoid multi-spin effects and to measure only the intermolecular stacking interactions...
November 7, 2018: RNA
Peter Y Wang, Alec N Sexton, William J Culligan, Matthew D Simon
Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil...
November 2, 2018: RNA
Jessica Döring, Thomas Hurek
Branchpoints in RNA templates are highly mutagenic, but it is not known yet whether this also applies to branchpoints in DNA templates. Here, we report how nucleic acid polymerases replicate a 2',5'-branched DNA (bDNA) molecule. We constructed long-chained bDNA templates containing a branch guanosine and T7 promoters at both arms by splinted-ligation. Quantitative real-time PCR analysis was used to investigate whether a branchpoint blocks DNA synthesis from the two arms in the same manner. We find that the blocking effect of a branchpoint is arm-specific...
October 25, 2018: RNA
Paul Yourik, Ryan T Fuchs, Megumu Mabuchi, Jennifer L Curcuru, G Brett Robb
Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences...
October 22, 2018: RNA
Witold Andrałojć, Magdalena Małgowska, Joanna Sarzyńska, Karol Pasternak, Kamil Szpotkowski, Ryszard Kierzek, Zofia Gdaniec
Uridine tetrads (U-tetrads) are a structural element encountered in RNA G-quadruplexes, for example, in the structures formed by the biologically relevant human telomeric repeat RNA. For these molecules, an unexpectedly strong stabilizing influence of a U-tetrad forming at the 3' terminus of a quadruplex was reported. Here we present the high resolution solution NMR structure of the r(UGGUGGU)4 quadruplex which, in our opinion, provides an explanation for this stabilization. Our structure features a distinctive, abrupt chain reversal just prior to the 3' uridine tetrad...
October 19, 2018: RNA
David Mitchell, Andrew J Renda, Catherine A Douds, Paul Babitzke, Sarah M Assmann, Philip C Bevilacqua
Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules, ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells...
October 19, 2018: RNA
Thilo Hetzke, Marc Vogel, Dnyaneshwar B Gophane, Julia E Weigand, Beatrix Suess, Snorri Th Sigurdsson, Thomas F Prisner
The tetracycline-binding RNA aptamer (TC-aptamer) is a synthetic riboswitch that binds the antibiotic tetracycline (TC) with exceptionally high affinity. Although a crystal structure exists of the TC-bound state, little is known about the conformational dynamics and changes upon ligand binding. In this study, pulsed electron paramagnetic resonance techniques for measuring distances (PELDOR) in combination with rigid nitroxide spin labels (Çm spin label), was used to investigate the conformational flexibility of the TC aptamer in the presence and absence of TC at different Mg2+ concentrations...
October 18, 2018: RNA
Nobuhiko Kamoshita, Shin-Ichi Tominaga
The translation of capsid proteins of Plautia stali intestine virus (PSIV), encoded in its second open reading frame (ORF2), is directed by an internal ribosomal entry site (IRES) located in the intergenic region (IGR). Owing to the specific properties of PSIV IGR in terms of nucleotide length and frame organization, capsid proteins are also translated via stop codon readthrough in mammalian cultured cells as an extension of translation from the first ORF (ORF1) and IGR. To delineate stop codon readthrough in PSIV, we determined requirements of cis-acting elements, through a molecular genetic approach applied in both cell-free translation systems and cultured cells...
October 18, 2018: RNA
Gracjan Patryk Michlewski, Javier F Caceres
MicroRNAs (miRNAs) are important regulators of gene expression that bind complementary target mRNAs and repress their expression. Precursor miRNA molecules undergo nuclear and cytoplasmic processing events, carried out by the endoribonucleases, DROSHA and DICER, respectively, to produce mature miRNAs that are loaded onto the RISC (RNA-induced silencing complex) to exert their biological function. Regulation of mature miRNA levels is critical in development, differentiation and disease, as demonstrated by multiple levels of control during their biogenesis cascade...
October 17, 2018: RNA
Lin Huang, Saira Ashraf, David M J Lilley
A recent study has shown that archaeal L7Ae binds to a putative k-turn structure in the 5'-leader of the mRNA of its structural gene to regulate translation. To function as a regulator the RNA should be unstructured in the absence of protein, but it should adopt a k-turn-containing stem-loop on binding L7Ae. Sequence analysis of UTR sequences indicates that their k-turn elements will be unable to fold in the absence of L7Ae, and we have demonstrated this experimentally in solution using FRET for the Archeoglobus fulgidus sequence...
October 16, 2018: RNA
Alexa A Lampasona, Kevin Czaplinski
Cell migration requires a complicated network of structural and regulatory proteins. Changes in cellular motility can impact migration as a result of cell-type or developmental stage regulated expression of critical motility genes. Hnrnpab is a conserved RNA binding protein found as two isoforms produced by alternative splicing. Its expression is enriched in the sub-ventricular zone (SVZ) and the rostral migratory stream within the brain, suggesting possible support of the migration of neural progenitor cells in this region...
October 12, 2018: RNA
Valerie Blanc, Yan Xie, Susan Kennedy, Jesse D Riordan, Deborah C Rubin, Blair B Madison, Jason C Mills, Joseph H Nadeau, Nicholas O Davidson
Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in-vitro RNA editing, A1cf-/- mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression...
October 11, 2018: RNA
Tomasz Krzywkowski, Malte Kühnemund, Mats Nilsson
Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1 DNA ligase) and T4RNA ligase 2 (T4Rnl2) on RNA...
October 11, 2018: RNA
Svetlana Deryusheva, Joseph G Gall
Posttranscriptional modifications of rRNA occur in the nucleolus where rRNA modification guide RNAs, or snoRNAs, concentrate. On the other hand, scaRNAs, the modification guide RNAs for spliceosomal snRNAs, concentrate in the Cajal body (CB). It is generally assumed, therefore, that snRNAs must accumulate in CBs to be modified by scaRNAs. Here we demonstrate that the evidence for the latter postulate is not consistent. In the nucleus, scaRNA localization is not limited to CBs. Furthermore, canonical scaRNAs can modify rRNAs...
October 9, 2018: RNA
Ruben M Atilho, Kevin R Perkins, Ronald R Breaker
Many bacteria use FMN (flavin mononucleotide) riboswitches to control the expression of genes responsible for the biosynthesis and transport of this enzyme cofactor or its precursor, riboflavin. Rare variants of FMN riboswitches found in strains of Clostridium difficile and some other bacteria typically control the expression of proteins annotated as transporters, including multidrug efflux pumps. These RNAs no longer recognize FMN, and differ from the original riboswitch consensus sequence at nucleotide positions normally involved in binding of the ribityl and phosphate moieties of the cofactor...
October 4, 2018: RNA
Kamil Kobylecki, Karolina Drazkowska, Tomasz M Kulinski, Andrzej Dziembowski, Rafal Tomecki
Pre-rRNA processing generates mature 18S, 5.8S and 28S/25S rRNAs through multistage removal of surrounding 5'-ETS/3'-ETS and intervening ITS1/ITS2 segments. Endonucleolytic activities release by-products, which need to be eliminated. Here, we investigated the interplay of exosome-associated 3'-5' exonucleases DIS3 and RRP6 in rRNA processing and by-product elimination in human cells. In agreement with previous reports, we observed accumulation of 5.8S and 18S precursors upon dysfunction of these enzymes. However, none of these phenotypes was so pronounced as previously overlooked accumulation of short RNA species derived from 5'-ETS (01/A'-A0), in cells with non-functional DIS3...
September 28, 2018: RNA
Frank A Tedeschi, Sara C Cloutier, Elizabeth J Tran, Eckhard Jankowsky
The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and lncRNA function. It is not understood how Dbp2p performs these seemingly unrelated biological roles. An important step towards addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using UV-crosslinking, denaturing tandem affinity purification, and Next Generation Sequencing...
September 27, 2018: RNA
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