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Nancy Laterreur, Bruno Lemieux, Hannah Neumann, Jean-Christophe Berger-Dancause, Daniel Lafontaine, Raymund J Wellinger
Telomerases are ribonucleoprotein (RNP) enzymes that are related to reverse transcriptases. While they maintain genome stability, their composition varies significantly between species. Yeast telomerase RNPs contain an RNA that is comparatively large and its overall folding shows long helical segments with distal functional parts. Here we investigated the essential stem IVc module of the budding yeast telomerase RNA, called Tlc1. The distal part of stem IVc includes a conserved sequence element CS2a and structurally conserved features to which bind the Pop1/Pop6/Pop7 proteins and which together function analogously to the P3 domains of the RNase P/MRP RNPs...
May 18, 2018: RNA
Jiansen Jiang, Yaqiang Wang, Lukas Sušac, Henry Chan, Ritwika Basu, Z Hong Zhou, Juli Feigon
Telomerase is an RNA-protein complex (RNP) that extends telomeric DNA at the 3' ends of chromosomes using its telomerase reverse transcriptase (TERT) and integral template-containing telomerase RNA (TER). Its activity is a critical determinant of human health, affecting aging, cancer, and stem cell renewal. Lack of atomic models of telomerase, particularly one with DNA bound, has limited our mechanistic understanding of telomeric DNA repeat synthesis. We report the 4.8 Å resolution cryoelectron microscopy structure of active Tetrahymena telomerase bound to telomeric DNA...
May 17, 2018: Cell
Baskar Bakthavachalu, Joern Huelsmeier, Indulekha P Sudhakaran, Jens Hillebrand, Amanjot Singh, Arnas Petrauskas, Devasena Thiagarajan, M Sankaranarayanan, Laura Mizoue, Eric N Anderson, Udai Bhan Pandey, Eric Ross, K VijayRaghavan, Roy Parker, Mani Ramaswami
Human Ataxin-2 is implicated in the cause and progression of amyotrophic lateral sclerosis (ALS) and type 2 spinocerebellar ataxia (SCA-2). In Drosophila, a conserved atx2 gene is essential for animal survival as well as for normal RNP-granule assembly, translational control, and long-term habituation. Like its human homolog, Drosophila Ataxin-2 (Atx2) contains polyQ repeats and additional intrinsically disordered regions (IDRs). We demonstrate that Atx2 IDRs, which are capable of mediating liquid-liquid phase transitions in vitro, are essential for efficient formation of neuronal mRNP assemblies in vivo...
May 16, 2018: Neuron
Omaththage P Perera, Nathan S Little, Calvin A Pierce
Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA...
2018: PloS One
Qiang Wang, Paul A Cobine, Jeffrey J Coleman
The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation...
May 12, 2018: Fungal Genetics and Biology: FG & B
Harriet Prior, Lauren MacConnachie, Jose L Martinez, Georgina C B Nicholl, Asim A Beg
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals...
April 30, 2018: Journal of Visualized Experiments: JoVE
Shuo-Chien Ling
TAR DNA-binding protein-43 KDa (TDP-43) and fused in sarcoma (FUS) as the defining pathological hallmarks for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coupled with ALS-FTD-causing mutations in both genes, indicate that their dysfunctions damage the motor system and cognition. On the molecular level, TDP-43 and FUS participate in the biogenesis and metabolism of coding and noncoding RNAs as well as in the transport and translation of mRNAs as part of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules...
2018: Neural Plasticity
Jongoh Shin, Namil Lee, Suhyung Cho, Byung-Kwan Cho
Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects...
2018: Methods in Molecular Biology
Andrew J Modzelewski, Sean Chen, Brandon J Willis, K C Kent Lloyd, Joshua A Wood, Lin He
CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions...
June 2018: Nature Protocols
Zachary J Kartje, Christopher L Barkau, Kushal J Rohilla, Eman A Ageely, Keith T Gagnon
DNA substitutions in RNA can probe the importance of A-form struc-ture, 2'-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. Using this approach, we found that Cas9 bio-chemical activity tolerated significant substitution with DNA nucleo-tides in the CRISPR RNA (crRNA). Only minimal RNA content was needed in or near the seed region. Simultaneous substitution at all positions with predicted crRNA-Cas9 2'-hydroxyl contacts had no effect on enzyme activity. The trans-activating crRNA (tracrRNA) also tolerated over 50% substitution with DNA...
May 10, 2018: Biochemistry
M Penrad-Mobayed, C Perrin, D L'Hôte, V Contremoulins, J-A Lepesant, B Boizet-Bonhoure, F Poulat, X Baudin, R A Veitia
Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis...
May 8, 2018: Scientific Reports
Martin A Newman, Fei Ji, Sylvia E J Fischer, Anthony Anselmo, Ruslan I Sadreyev, Gary Ruvkun
RNAi pathways detect and silence foreign nucleic acids such as viruses as well as endogenous genes in many species. The phylogenetic profile across eukaryotes of proteins that mediate key steps in RNAi is correlated with the profiles of multiple mRNA splicing proteins and with intron number, suggesting that RNAi may surveil mRNA splicing to detect the divergent or absent introns of viruses. Here we examine the role of mRNA splicing in Caenorhabditis elegans RNAi . We found that viable null mutations in U1 and U2 small nuclear ribonucleic protein (snRNP)-specific splicing factor genes cause defects in RNAi...
May 8, 2018: Genes & Development
Jordan E Burke, Adam D Longhurst, Daria Merkurjev, Jade Sales-Lee, Beiduo Rao, James J Moresco, John R Yates, Jingyi Jessica Li, Hiten D Madhani
Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection of canonical and non-canonical events, including interrupted, recursive, and nested splicing. This application of statistical modeling uncovers independent roles for the size and position of the intron and the number of introns per transcript in substrate progression through the two catalytic stages...
May 3, 2018: Cell
C Eno, F Pelegri
During the early embryonic cell cycles, zebrafish germ plasm ribonucleoparticles (RNPs) gradually multimerize and become recruited to the forming furrows. RNPs multimerization occurs prior to and during furrow initiation, as forming aggregates move outward through their association with the tips of growing interphase astral microtubules. Germ plasm RNPs are also associated with short cortical F-actin. We show that, in embryos mutant for the cytoskeletal regulator mid1ip1L , germ plasm RNPs fail to become recruited to the furrow, accumulating instead at the periphery of the blastodisc...
May 3, 2018: Development
Zhen Liang, Kunling Chen, Yan Yan, Yi Zhang, Caixia Gao
Despite the great achievements in genome editing, accurately detecting mutations induced by sequence-specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome-edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site...
May 3, 2018: Plant Biotechnology Journal
Anna Perederina, Igor Berezin, Andrey S Krasilnikov
RNase P is a ubiquitous site-specific endoribonuclease primarily responsible for the maturation of tRNA. Throughout the three domains of life, the canonical form of RNase P is a ribonucleoprotein (RNP) built around a catalytic RNA. The core RNA is well conserved from bacteria to eukaryotes, whereas the protein parts vary significantly. The most complex and the least understood form of RNase P is found in eukaryotes, where multiple essential proteins playing largely unknown roles constitute the bulk of the enzyme...
May 2, 2018: Nucleic Acids Research
Zunlong Ke, Joshua D Strauss, Cheri M Hampton, Melinda A Brindley, Rebecca S Dillard, Fredrick Leon, Kristen M Lamb, Richard K Plemper, Elizabeth R Wright
Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles...
April 30, 2018: Nature Communications
Lorenzo Brunetti, Michael C Gundry, Ayumi Kitano, Daisuke Nakada, Margaret A Goodell
Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments...
April 10, 2018: Journal of Visualized Experiments: JoVE
Thi Hoang Duong Nguyen, Jane Tam, Robert A Wu, Basil J Greber, Daniel Toso, Eva Nogales, Kathleen Collins
The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP)...
April 25, 2018: Nature
Junjie Li, Jiangang Xie, Shanshou Liu, Xiao Li, Dongliang Zhang, Xianqi Wang, Jinquan Jiang, Wei Hu, Yuan Zhang, Boquan Jin, Ran Zhuang, Wen Yin
ADAR1 (adenosine deaminase acting on double-stranded RNA 1) is an RNA-editing enzyme that mediates adenosine-to-inosine RNA editing events, an important post-transcriptional modification mechanism that can alter the coding properties of mRNA or regulate microRNA biogenesis. ADAR1 also regulates the innate immune response. Here, we have demonstrated that ADAR1 expression increased in LPS-stimulated macrophages. Silencing ADAR1 by using small interfering RNA in macrophages resulted in the pronounced polarization of macrophages to M1, whereas ADAR1 overexpression promoted M2 polarization, which indicated that ADAR1 can inhibit macrophage hyperpolarization and prevent immune hyperactivity...
April 25, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
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