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Marius van den Beek, Bruno da Silva, Juliette Pouch, Mohammed El Amine Ali Chaouche, Clément Carré, Christophe Antoniewski
piRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation of heritable mutations caused by their transposition in the genome. It is not clear whether the piRNA pathway plays a functional role in adult, non-gonadal tissues in Drosophila melanogaster. To address this question, we first analyzed the small RNA content of adult Drosophila melanogaster heads. We found that varying amount of piRNA-sized, ping-pong positive molecules in heads correlates with contamination by gonadal tissue during RNA extraction, suggesting that most of piRNAs detected in head sequencing libraries originate from gonads...
September 14, 2018: RNA
Howard Gamper, Ya-Ming Hou
Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to non-ribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown...
September 14, 2018: RNA
Joanna Kwiatkowska, Zuzanna Wroblewska, Kenneth A Johnson, Mikolaj Olejniczak
MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg2+ ions in Escherichia coli. MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I, sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, eptB and ygdQ by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products...
September 14, 2018: RNA
Bret R Miller, Tianqi Wei, Christopher J Fields, Peike Sheng, Mingyi Xie
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive Northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of Northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive Northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern)...
September 10, 2018: RNA
Daniel P Reich, Brenda L Bass
Complementary sequences in cellular transcripts base-pair to form double-stranded RNA (dsRNA) structures. Because transposon-derived repeats often give rise to self-complementary sequences, dsRNA structures are prevalent in eukaryotic genomes, typically occurring in gene introns and untranslated regions (UTRs). However, the regulatory impact of double-stranded structures within genes is not fully understood. We used three independent methods to define loci in Caenorhabditis elegans predicted to form dsRNA and correlated these structures with patterns of gene expression, gene essentiality, and genome organization...
September 6, 2018: RNA
Wen-Xia He, Min Wu, Zhen Liu, Zhi Li, Yang Wang, Jian Zhou, Peng Yu, Xiao-Juan Zhang, Li Zhou, Jian-Fang Gui
Stem-loop binding protein (SLBP) has been revealed to bind to the stem-loop of replication-dependent histone mRNAs and to be required for replication-dependent histone mRNA metabolism in mammals. Zebrafish possesses two slbps and slbp1 is required for retinal neurogenesis. However, the detailed expression and function of slbp2 in zebrafish is still unknown. In this study, we first identified zebrafish slbp2 as an oocyte-specific maternal factor and then generated maternal-zygotic slbp2 F3 homozygous mutant (MZslbp2Δ4-/-) by CRISPR/Cas9...
September 5, 2018: RNA
Susan J Schroeder
The revolution in sequencing technology demands new tools to interpret the genetic code. As in vivo transcriptome-wide chemical probing techniques advance, new challenges emerge in the RNA folding problem. The emphasis on one sequence folding into a single minimum free energy structure is fading as a new focus develops on generating RNA structural ensembles and identifying functional structural features in ensembles. This review describes an efficient combinatorially complete method and three free energy-minimization approaches to predicting RNA structures with more than one functional fold...
August 24, 2018: RNA
Beáta Jády, Amandine Ketele, Tamás Kiss
Cytoplasmic localization, stability and translation of mRNAs are controlled by their dynamic association of numerous mRNA-binding (mRNP) proteins, including cold shock domain- (CSD)-containing proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins. Here, we demonstrate that the most abundant human mRNP protein, the CSD-containing Y-box-binding protein 1 (YBX1), the closely related YBX3 protein and other mRNP proteins, such as SRSF1, SRSF2, SRSF3, hnRNP A1 and H, specifically and efficiently interact with overlapping sets of mitochondrial tRNAs (mt tRNAs)...
August 23, 2018: RNA
E Han Dao, Frédéric Poitevin, Raymond G Sierra, Cornelius Gati, Yashas Rao, Halil Ibrahim Ciftci, Fulya Akşit, Alex McGurk, Trevor Obrinski, Paul Mgbam, Brandon Hayes, Casper DE Lichtenberg, Fatima Pardo-Avila, Nicholas Corsepius, Lindsey Zhang, Matthew H Seaberg, Mark S Hunter, Mengling Liang, Jason E Koglin, Soichi Wakatsuki, Hasan Demirci
The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New lightsources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3...
August 23, 2018: RNA
Peter Schäfer, Christian Tüting, Lars Schönemann, Uwe Kühn, Thomas Treiber, Nora Treiber, Christian Ihling, Anne Graber, Walter Keller, Gunter Meister, Andrea Sinz, Elmar Wahle
Cleavage factor II (CF II) is a poorly characterized component of the multi-protein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11...
August 23, 2018: RNA
Min Jia, Heinz Gut, Jeffrey A Chao
The IMP family of RNA binding proteins, also named as insulin-like growth factor 2 (IGF2) mRNA-binding proteins (IGF2BPs), are highly conserved RNA regulators that are involved in many RNA processing stages, including mRNA stability, localization and translation. There are three paralogs in the IMP family, IMP1-3, in mammals that all adopt the same domain arrangement with two RNA recognition motifs (RRM) in the N-terminus and four KH domains in the C-terminus. Here, we report the structure and biochemical characterization of IMP3 RRM12 and its complex with two short RNAs...
August 22, 2018: RNA
Schraga Schwartz
Following synthesis, RNA can be modified with over 100 chemically distinct modifications. Recently, two studies - one by our group - developed conceptually similar approaches to globally map N1-methyladenosine (m1A) at single nucleotide resolution. Surprisingly, the studies diverged quite substantially in their estimates of the abundance, whereabouts and stoichiometry of צ1A within internal sites in cytosolic mRNAs: One study reported it to be a very rare modification, present at very low stoichiometries, and invariably catalyzed by TRMT6/61A...
August 21, 2018: RNA
Xushen Xiong, Xiaoyu Li, Kun Wang, Chengqi Yi
N 1 -methyladenosine was recently reported to be a chemical modification in mRNA. However, while we identified hundreds of m1 A sites in the human transcriptome (Li et al., 2017), Safra et al. detected only 9 sites in the cytosolic and mitochondrial mRNAs (Safra et al., 2017). Herein, we provide additional evidences that hundreds of m1 A sites are present in the human transcriptome. Moreover, we show that both the improper bioinformatic tools and the poor quality of sequencing data in the Safra et al. study lead to the failure in identifying the majority of the m1 A sites...
August 21, 2018: RNA
Sameer Dixit, Julius Lukes
MRP1/2 is a heteromeric protein complex that functions in the trypanosomatid mitochondrion as part of the RNA editing machinery, which facilitates multiple targeted insertions and deletions of uridines. MRP1/2 was shown to interact with MRB8170, which initiates RNA editing by marking pre-edited mRNAs, while TbRGG2 is required for its efficient progression on pan-edited mRNAs. Both MRP1/2 and TbRGG2 are capable of modulating RNA-RNA interactions in vitro. As determined by using iCLIP and RIP-qPCR, RNAs bound to MRP1/2 are characterized and compared with those associated with MRB8170 and TbRGG2...
August 17, 2018: RNA
Akiyoshi Nakamura, Daole Wang, Yasuo Komatsu
The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3'-5' direction onto the 5'-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the -1 position (G-1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first, we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis...
August 15, 2018: RNA
Neil A White, Minako Sumita, Victor E Marquez, Charles G Hoogstraten
In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a North-methanocarbacytidine modification that prevents fluctuations to C2'-endo conformations while maintaining an intact 2'-hydroxyl nucleophile...
August 15, 2018: RNA
Jeffrey Zuber, B Joseph Cabral, Iain McFadyen, David M Mauger, David H Mathews
RNA secondary structure prediction is often used to develop hypotheses about structure-function relationships for newly discovered RNA sequences, to identify unknown functional RNAs, and to design sequences. Secondary structure prediction methods typically use a thermodynamic model that estimates the free energy change of possible structures based on a set of nearest neighbor parameters. These parameters were derived from optical melting experiments of small model oligonucleotides. This work aims to better understand the precision of structure prediction...
August 13, 2018: RNA
Kristina Keidel, Fabian Amman, Ilona Bibova, Jakub Drzmisek, Vladimir Benes, David Hot, Branislav Vecerek
Bordetella pertussis is the causative agent of human whooping cough (pertussis), a highly contagious respiratory disease which despite massive vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression in this re-emerging pathogen. High through-put RNA sequencing revealed hundreds of putative non-coding RNAs including the RgtA sRNA...
August 10, 2018: RNA
Stanislav Bellaousov, Mohammad Kayedkhordeh, Raymond J Peterson, David H Mathews
Nanoscale nucleic acids can be designed to be nano-machines, pharmaceuticals, or probes for detecting pathogens and other molecules. RNA secondary structures can form the basis of self-assembling nanostructures. There are only four natural RNA bases, therefore it can be difficult to design sequences that fold to a single, specified structure because many other structures are often possible for a given sequence. One approach taken by state-of-the-art sequence design methods is to select sequences that fold to the specified input structure using stochastic, iterative refinement...
August 10, 2018: RNA
Tong Zhou, Xueying Xie, Musheng Li, Junchao Shi, Jin Zhou, Kenneth Knox, Ting Wang, Qi Chen, Wanjun Gu
Circular RNAs (circRNAs) are a novel class of regulatory RNAs. Here, we present a comprehensive investigation of circRNA expression profiles across 11 tissues and 4 developmental stages in rats, along with cross-species analyses in humans and mice. Although the expression of circRNAs is positively correlated with that of cognate mRNAs, highly expressed genes tend to splice a larger fraction of circular transcripts. Moreover, circRNAs exhibit higher tissue specificity than cognate mRNAs. Intriguingly, while we observed a monotonic increase of circRNA abundance with age in the rat brain, we further discovered a dynamic, age-dependent pattern of circRNA expression in the testes that is characterized by a dramatic increase with advancing stages of sexual maturity and a decrease with aging...
August 9, 2018: RNA
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