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Jakub Stanislaw Nowak, Fruzsina Hobor, Angela Downie Ruiz Velasco, Nila Roy Choudhury, Gregory Heikel, Alastair Kerr, Andres Ramos, Gracjan Michlewski
Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production...
November 23, 2016: RNA
Brice Felden, Luc Paillard
Bacterial and eukaryotic immunity RNA clusters share unexpected strong structural and functional resemblances in fighting DNA intruders.
November 23, 2016: RNA
Megumi Shigematsu, Yohei Kirino
Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived non-coding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the -1 position (G-1) of its 5'-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method which can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G-1, U-1, A-1, or C-1 or lacking the -1 nucleotide (starting from G1)...
November 22, 2016: RNA
Byron Lee, Ryan Alexander Flynn, Anastasia Kadina, Jimmy K Guo, Eric T Kool, Howard Y Chang
Recent advances in SHAPE technology have converted the classic primer extension method to next generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N3 and 1M7 reagents respectively, are methods that claim to measure in vivo structure with high-throughput sequencing. However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay...
November 22, 2016: RNA
Jean Lehmann
The catalytic site of most enzymes can efficiently handle only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced-fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction...
November 22, 2016: RNA
Nils Schneider, Matthias Meier
Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands...
November 22, 2016: RNA
Claire V Crowther, Laura E Jones, Jessica N Morelli, Eric M Mastrogiacomo, Claire Porterfield, Jessica L Kent, Martin J Serra
53 RNA duplexes containing two single nucleotide bulge loops were optically melted in 1M NaCl in order to determine the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each duplex. Because of the large number of possible combinations and lack of sequence effects observed previously, we limited our initial investigation to adenosine bulges, the most common naturally occurring bulge. For example, the following duplexes were investigated: 5' GGCAXYAGGC 5' GGCAXY GCC 5' GGC XYAGCC CCG YX CCG CCG YXACGG CCGAXY CGG The identity of XY (where XY are Watson Crick base pairs) and the total number of base pairs in the terminal and central stems were varied...
November 21, 2016: RNA
Rimpi Khurana, Glory Ranches, Simon Schafferer, Melanie Lukasser, Michael Rudnicki, Gert Mayer, Alexander Hüttenhofer
In chronic kidney disease (CKD), the decline in glomerular filtration rate is associated with increased morbidity and mortality and thus poses a major challenge for healthcare systems. While the contribution of tissue-derived miRNAs and mRNAs to CKD progression has extensively been studied, little is known about the role of urinary exosomes and their association with CKD. Exosomes are small, membrane-derived endocytic vesicles that contribute to cell-to-cell communication and are present in various body fluids, such as blood or urine...
November 21, 2016: RNA
Martino Colombo, Evangelos D Karousis, Joël Bourquin, Rémy Bruggmann, Oliver Mühlemann
Besides degrading aberrant mRNAs that harbor a premature translation termination codon (PTC), nonsense-mediated mRNA decay (NMD) also targets many seemingly "normal" mRNAs that encode for full-length proteins. To identify a bona fide set of such endogenous NMD targets in human cells, we applied a meta-analysis approach in which we combined transcriptome profiling of knockdowns and rescues of the three NMD factors UPF1, SMG6 and SMG7. We provide evidence that this combinatorial approach identifies NMD-targeted transcripts more reliably than previous attempts that focused on inactivation of single NMD factors...
November 18, 2016: RNA
Edward M Kennedy, Anand V R Kornepati, Hal P Bogerd, Bryan R Cullen
While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two co-factors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR...
November 11, 2016: RNA
William A Cantara, Joshua Hatterschide, Weixin Wu, Karin Musier-Forsyth
Chemical and enzymatic probing of RNA secondary structure and RNA/protein interactions provides the basis for understanding the functions of structured RNAs. However, the ability to rapidly perform such experiments using capillary electrophoresis has been hampered by relatively labor-intensive data analysis software. While these computationally robust programs have been shown to calculate residue-specific reactivities to a high degree of accuracy, they often require time-consuming manual intervention and lack the ability to be easily modified by users...
November 7, 2016: RNA
Yuedong Yang, Xiaomei Li, Huiying Zhao, Jian Zhan, Jihua Wang, Yaoqi Zhou
As most RNA structures are elusive to structure determination, obtaining solvent accessible surface areas (ASA) of nucleotides in an RNA structure is an important first step to characterize potential functional sites and core structural regions. Here, we developed RNAsnap, the first machine-learning method trained on protein-bound RNA structures for solvent accessibility prediction. Built on sequence profiles from multiple sequence alignment (RNAsnap-prof), the method provided robust prediction in five-fold cross-validation and an independent test (Pearson correlation coefficients, r, between predicted and actual ASA values are 0...
November 2, 2016: RNA
Kalle J Hanhijärvi, Gabija Ziedaite, Dennis H Bamford, Edward Hæggström, Minna M Poranen
Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage phi6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative-strands are synthesized...
November 1, 2016: RNA
Andrey Krokhotin, Anthony M Mustoe, Kevin M Weeks, Nikolay V Dokholyan
Many RNA molecules fold into complex secondary and tertiary structures that play critical roles in biological function. Among the best-established methods for examining RNA structure are chemical probing experiments, which can report on local nucleotide structure in a concise and extensible manner. While probing data are highly useful for inferring overall RNA secondary structure, these data do not directly measure through-space base pairing interactions. We recently introduced an approach for single-molecule correlated chemical probing with dimethyl sulfate (DMS) that measures RNA interaction groups by mutational profiling (RING-MaP)...
November 1, 2016: RNA
Nicholas Vrettos, Manolis Maragkakis, Panagiotis Alexiou, Zissimos Mourelatos
PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Cell lines that endogenously express piRNAs and PIWI proteins are an invaluable complement to whole-organism studies as they facilitate molecular and biochemical investigations of piRNAs. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi...
October 27, 2016: RNA
Fan Yang, Peter Hsu, Susan D Lee, Wen Yang, Derick Hoskinson, Weihao Xu, Claire Moore, Gabriele Varani
3'-end processing of pre-mRNAs prior to packaging and export to the cytoplasm of the mature transcript is a highly regulated process executed by several tens of protein factors that recognize poorly conserved RNA signals. Among them is Pcf11, a highly conserved, multi-domain protein that links transcriptional elongation, 3'-end processing and transcription termination. Here we report the structure and biochemical function of Pcf11's C-terminal domain, which is conserved from yeast to humans. We identify a novel zinc finger fold, resembling a trillium flower...
October 25, 2016: RNA
Hanqian Mao, Hannah E Brown, Debra L Silver
The exon junction complex (EJC) is a multi-protein complex integral to RNA metabolism. Biochemistry and genetic studies have concluded that the EJC is composed of 4 core proteins, MAGOH, EIF4A3, RBM8A, and CASC3. Yet recent studies in Drosophila indicate divergent functions for Barentsz, the mammalian Casc3 ortholog, raising the question as to whether CASC3 is a constitutive component of the EJC. This issue remains poorly understood, particularly in an in vivo mammalian context. We previously found that haploinsufficiency for Magoh, Eif4a3, or Rbm8a disrupts neuronal viability and neural progenitor proliferation, resulting in severe microcephaly...
October 25, 2016: RNA
Keisuke Shoji, Yutaka Suzuki, Sumio Sugano, Toru Shimada, Susumu Katsuma
PIWI-interacting RNAs (piRNAs) play essential roles in the defense system against selfish elements in animal germ line cells by cooperating with PIWI proteins. A subset of piRNAs is predicted to be generated via the "ping-pong" cascade, which is mainly controlled by two different PIWI proteins. Here we established a cell-based artificial piRNA production system using a silkworm ovarian cultured cell line that is believed to possess a complete piRNA pathway. In addition, we took advantage of a unique silkworm sex-determining one-to-one ping-pong piRNA pair, which enabled us to precisely monitor the behaviour of individual artificial piRNAs...
October 24, 2016: RNA
Rei Yoshimoto, Daisuke Kaida, Masaaki Furuno, A Maxwell Burroughs, Shohei Noma, Harukazu Suzuki, Yumi Kawamura, Yoshihide Hayashizaki, Akila Mayeda, Minoru Yoshida
Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest...
October 17, 2016: RNA
Cindo O Nicholson, Matthew B Friedersdorf, Jack D Keene
RNA-binding proteins (RBPs) and non-coding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site, and quantifies binding to whole transcripts...
October 14, 2016: RNA
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