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https://www.readbyqxmd.com/read/27902801/achieving-plant-crispr-targeting-that-limits-off-target-effects
#1
Jeffrey D Wolt, Kan Wang, Dipali Sashital, Carolyn J Lawrence-Dill
The CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein) has been used to generate targeted changes for direct modification of endogenous genes in an increasing number of plant species; but development of plant genome editing has not yet fully considered potential off-target mismatches that may lead to unintended changes within the genome. Assessing the specificity of CRISPR-Cas9 for increasing editing efficiency as well as the potential for unanticipated downstream effects from off-target mutations is an important regulatory consideration for agricultural applications...
November 2016: Plant Genome
https://www.readbyqxmd.com/read/27892924/the-pattern-recognition-receptor-core-of-solanaceae-detects-bacterial-cold-shock-protein
#2
Lei Wang, Markus Albert, Elias Einig, Ursula Fürst, Damaris Krust, Georg Felix
Plants and animals recognize microbial invaders by detecting microbe-associated molecular patterns (MAMPs) by cell surface receptors. Many plant species of the Solanaceae family detect the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), represented by the peptide csp22, as a MAMP. Here, we exploited the natural variation in csp22 perception observed between cultivated tomato (Solanum lycopersicum) and Solanum pennellii to map and identify the leucine-rich repeat (LRR) receptor kinase CORE (cold shock protein receptor) of tomato as the specific, high-affinity receptor site for csp22...
November 28, 2016: Nature Plants
https://www.readbyqxmd.com/read/27882870/large-scale-remodeling-of-a-repressed-exon-ribonucleoprotein-to-an-exon-definition-complex-active-for-splicing
#3
Somsakul Pop Wongpalee, Ajay Vashisht, Shalini Sharma, Darryl Chui, James A Wohlschlegel, Douglas L Black
Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition...
November 24, 2016: ELife
https://www.readbyqxmd.com/read/27871973/par-clip-and-streamlined-small-rna-cdna-library-preparation-protocol-for-the-identification-of-rna-binding-protein-target-sites
#4
Daniel Benhalevy, Hannah L McFarland, Aishe A Sarshad, Markus Hafner
The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale...
November 18, 2016: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/27870764/anti-ro-and-concomitant-anti-la-autoantibodies-strongly-associated-with-anti-oxldl-or-anti-phospholipid-antibody-in-systemic-lupus-erythematosus
#5
Biji Theyilamannil Kurien, James Fesmire, Camille Jean Anderson, Robert Hal Scofield
BACKGROUND: Premature atherosclerosis is observed in systemic lupus erythematosus (SLE). Oxidative modification of LDL is associated with atherosclerotic plaque formation. OBJECTIVES: We hypothesized that anti-oxidized LDL (oxLDL) and anti-phospholipid (APL) in SLE sera would segregate with specific antibody subsets, and that anti-oxLDL antibodies will linger in circulation over an extended period. PATIENTS AND METHODS: Sixty-seven SLE and control subjects and two SLE subjects with sera collected longitudinally for 13 years were tested for anti-oxLDL and IgG/IgM/IgA APL antibodies...
December 2016: Journal of Clinical Rheumatology: Practical Reports on Rheumatic & Musculoskeletal Diseases
https://www.readbyqxmd.com/read/27864990/the-clinical-relevance-of-anti-fibrillarin-anti-u3-rnp-autoantibodies-in-systemic-sclerosis
#6
F Tall, M Dechomet, S Riviere, V Cottin, E Ballot, K P Tiev, R Montin, C Morin, Y Chantran, C Grange, D Jullien, J Ninet, P Chretien, J Cabane, N Fabien, C Johanet
OBJECTIVES: Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several antinuclear autoantibodies useful to diagnosis and prognosis. The aim of the present multicentric study was to determine the clinical relevance of anti-fibrillarin autoantibodies (AFA) in patients with SSc. METHODS: The clinical features of 37 SSc patients positive for AFA (AFA+) and 139 SSc patients without AFA (AFA-) were collected retrospectively from medical records to enable a comparison between AFA- and AFA+ patients...
November 16, 2016: Scandinavian Journal of Immunology
https://www.readbyqxmd.com/read/27856639/the-atypical-dual-specificity-phosphatase-hyvh1-associates-with-multiple-ribonucleoprotein-particles
#7
Qiudi Geng, Besa Xhabija, Colleen Knuckle, Christopher A Bonham, Panayiotis O Vacratsis
Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12) is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress induced cell death, while studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1 associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation...
November 17, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27852926/solution-structure-of-the-5-terminal-hairpin-of-the-7sk-small-nuclear-rna
#8
Sarah Bourbigot, Anne-Catherine Dock-Bregeon, Pascal Eberling, Jérôme Coutant, Bruno Kieffer, Isabelle Lebars
The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat...
December 2016: RNA
https://www.readbyqxmd.com/read/27845164/enhanced-crispr-cas9-mediated-precise-genome-editing-by-improved-design-and-delivery-of-grna-cas9-nuclease-and-donor-dna
#9
Xiquan Liang, Jason Potter, Shantanu Kumar, Namritha Ravinder, Jonathan D Chesnut
While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA...
November 11, 2016: Journal of Biotechnology
https://www.readbyqxmd.com/read/27819531/toward-an-understanding-of-regulating-cajal-body-activity-by-protein-modification
#10
Michael D Hebert, Aaron R Poole
The biogenesis of small nuclear ribonucleoproteins (snRNPs), small Cajal body-specific RNPs (scaRNPs), small nucleolar RNPs (snoRNPs) and the telomerase RNP involves Cajal bodies (CBs). Although many components enriched in the CB contain post-translational modifications (PTMs), little is known about how these modifications impact individual protein function within the CB and, in concert with other modified factors, collectively regulate CB activity. Since all components of the CB also reside in other cellular locations, it is also important that we understand how PTMs affect the subcellular localization of CB components...
October 7, 2016: RNA Biology
https://www.readbyqxmd.com/read/27810925/cd72-negatively-regulates-b-lymphocyte-responses-to-the-lupus-related-endogenous-toll-like-receptor-7-ligand-sm-rnp
#11
Chizuru Akatsu, Kenro Shinagawa, Nobutaka Numoto, Zhihong Liu, Ayse Konuskan Ucar, Mohammad Aslam, Shirly Phoon, Takahiro Adachi, Koji Furukawa, Nobutoshi Ito, Takeshi Tsubata
Toll-like receptor 7 (TLR7) plays an essential role in development of systemic lupus erythematosus by co-stimulating B cells reactive to the endogenous TLR7 ligand Sm/ribonucleoprotein (RNP), a crucial lupus self-antigen. However, how the TLR7-mediated autoimmune response is regulated is not yet known. In this study, we demonstrate that CD72, an inhibitory B cell co-receptor known to prevent development of lupus, recognizes Sm/RNP at the extracellular C-type lectin-like domain (CTLD) and specifically inhibits B cell response to Sm/RNP...
November 14, 2016: Journal of Experimental Medicine
https://www.readbyqxmd.com/read/27795444/the-dual-roles-of-the-ha-segment-specific-noncoding-nucleotides-in-the-extended-duplex-region-of-the-influenza-a-virus-rna-promoter
#12
Jingfeng Wang, Jinghua Li, Lili Zhao, Mengmeng Cao, Tao Deng
: We recently reported that the segment-specific noncoding regions (NCRs) of the HA and NA segments are subtype-specific, varying significantly in sequence and length at both the 3' and 5' ends. Interestingly, we found that nucleotides "CC" at positions 13-14 at the 3' end and "GUG" at positions 14' -16' at the 5' end are absolutely conserved among all HA subtype-specific NCRs. These HA segment-specific NCR nucleotides are located in the extended duplex region of the viral RNA promoter...
October 26, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27791212/biophysical-characterization-of-organelle-based-rna-protein-liquid-phases-using-microfluidics
#13
Nicole Taylor, Shana Elbaum-Garfinkle, Nilesh Vaidya, Huaiying Zhang, Howard A Stone, Clifford P Brangwynne
Living cells contain numerous membrane-less RNA/protein (RNP) bodies that assemble by intracellular liquid-liquid phase separation. The properties of these condensed phase droplets are increasingly recognized as important in their physiological function within living cells, and also through the link to protein aggregation pathologies. However, techniques such as droplet coalescence analysis or standard microrheology do not always enable robust property measurements of model RNA/protein droplets in vitro. Here, we introduce a microfluidic platform that drives protein droplets into a single large phase, which facilitates viscosity measurements using passive microrheology and/or active two-phase flow analysis...
November 16, 2016: Soft Matter
https://www.readbyqxmd.com/read/27783956/highly-efficient-genome-editing-of-murine-and-human-hematopoietic-progenitor-cells-by-crispr-cas9
#14
Michael C Gundry, Lorenzo Brunetti, Angelique Lin, Allison E Mayle, Ayumi Kitano, Dimitrios Wagner, Joanne I Hsu, Kevin A Hoegenauer, Cliona M Rooney, Margaret A Goodell, Daisuke Nakada
Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%)...
October 25, 2016: Cell Reports
https://www.readbyqxmd.com/read/27769719/analysis-of-the-cleavage-mechanism-by-protein-only-rnase-p-using-precursor-trna-substrates-with-modifications-at-the-cleavage-site
#15
Dennis Walczyk, Markus Gößringer, Walter Rossmanith, Timofei S Zatsepin, Tatiana S Oretskaya, Roland K Hartmann
Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino or 2'-O-methyl substitutions...
October 18, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27733558/selection-free-genome-editing-of-the-sickle-mutation-in-human-adult-hematopoietic-stem-progenitor-cells
#16
Mark A DeWitt, Wendy Magis, Nicolas L Bray, Tianjiao Wang, Jennifer R Berman, Fabrizia Urbinati, Seok-Jin Heo, Therese Mitros, Denise P Muñoz, Dario Boffelli, Donald B Kohn, Mark C Walters, Dana Carroll, David I K Martin, Jacob E Corn
Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34(+) hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs...
October 12, 2016: Science Translational Medicine
https://www.readbyqxmd.com/read/27730127/functionality-of-in-vitro-reconstituted-group-ii-intron-rmint1-derived-ribonucleoprotein-particles
#17
Maria D Molina-Sánchez, Fernando M García-Rodríguez, Nicolás Toro
The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms...
2016: Frontiers in Molecular Biosciences
https://www.readbyqxmd.com/read/27727192/cross-and-co-packaging-of-retroviral-rnas-and-their-consequences
#18
Lizna M Ali, Tahir A Rizvi, Farah Mustafa
Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP) particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA) among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins...
October 11, 2016: Viruses
https://www.readbyqxmd.com/read/27726526/long-term-memory-consolidation-the-role-of-rna-binding-proteins-with-prion-like-domains
#19
Indulekha P Sudhakaran, Mani Ramaswami
Long-term and short-term memories differ primarily in the duration of their retention. At a molecular level, long-term memory (LTM) is distinguished from short-term memory (STM) by its requirement for new gene expression. In addition to transcription (nuclear gene expression) the translation of stored mRNAs is necessary for LTM formation. The mechanisms and functions for temporal and spatial regulation of mRNAs required for LTM is a major contemporary problem, of interest from molecular, cell biological, neurobiological and clinical perspectives...
October 11, 2016: RNA Biology
https://www.readbyqxmd.com/read/27726486/human-intron-encoded-aluaca-rnas-and-telomerase-rna-share-a-common-element-promoting-rna-accumulation
#20
Amandine Ketele, Tamás Kiss, Beáta E Jády
Mammalian cells express hundreds of intron-encoded box H/ACA RNAs which fold into a common hairpin-hinge-hairpin-tail structure, interact with four evolutionarily conserved proteins, dyskerin, Nop10, Nhp2 and Gar1, and function mainly in RNA pseudouridylation. The human telomerase H/ACA RNA (hTR) directs telomeric DNA synthesis and it carries a 5'-terminal domain encompassing the telomeric template sequence. The primary hTR transcript is synthesized from an independent gene by RNA polymerase II and undergoes 3' end processing controlled by the 3'-terminal H/ACA domain...
October 11, 2016: RNA Biology
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