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https://www.readbyqxmd.com/read/28431230/structural-basis-for-guide-rna-processing-and-seed-dependent-dna-targeting-by-crispr-cas12a
#1
Daan C Swarts, John van der Oost, Martin Jinek
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28429714/pif1-family-dna-helicases-suppress-r-loop-mediated-genome-instability-at-trna-genes
#2
Phong Lan Thao Tran, Thomas J Pohl, Chi-Fu Chen, Angela Chan, Sebastian Pott, Virginia A Zakian
Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. Rrm3 promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). We identify a new role for the Pif1 helicase: promotion of replication and suppression of DNA damage at tDNAs. Pif1 binds multiple tDNAs, and this binding is higher in rrm3Δ cells. Accumulation of replication intermediates and DNA damage at tDNAs is higher in pif1Δ rrm3Δ than in rrm3Δ cells. DNA damage at tDNAs in the absence of these helicases is suppressed by destabilizing R-loops while Pif1 and Rrm3 binding to tDNAs is increased upon R-loop stabilization...
April 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28418376/a-circrna-from-sepallata3-regulates-splicing-of-its-cognate-mrna-through-r-loop-formation
#3
Vanessa M Conn, Véronique Hugouvieux, Aditya Nayak, Stephanie A Conos, Giovanna Capovilla, Gökhan Cildir, Agnès Jourdain, Vinay Tergaonkar, Markus Schmid, Chloe Zubieta, Simon J Conn
Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life(1), yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family...
April 18, 2017: Nature Plants
https://www.readbyqxmd.com/read/28412545/dna-topoisomerase-1-prevents-r-loop-accumulation-to-modulate-auxin-regulated-root-development-in-rice
#4
Sarfraz Shafiq, Chunli Chen, Jing Yang, Lingling Cheng, Fei Ma, Emilie Widemann, Qianwen Sun
R-loop structures (RNA:DNA hybrids) have important functions in many biological processes, including transcriptional regulation and genome instability among diverse organisms. DNA Topoisomerase 1 (TOP1), an essential manipulator of DNA topology during RNA transcription and DNA replication processes, can prevent R-loop accumulation by removing the positive and negative DNA supercoiling that is made by RNA polymerases during transcription. TOP1 is required for plant development, but little is known about its function in preventing co-transcriptional R-loops in different plant biological processes...
April 12, 2017: Molecular Plant
https://www.readbyqxmd.com/read/28408437/the-myc-mrna-3-utr-couples-rna-polymerase-ii-function-to-glutamine-and-ribonucleotide-levels
#5
Francesca R Dejure, Nadine Royla, Steffi Herold, Jacqueline Kalb, Susanne Walz, Carsten P Ade, Guido Mastrobuoni, Jens T Vanselow, Andreas Schlosser, Elmar Wolf, Stefan Kempa, Martin Eilers
Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels...
April 13, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28406751/repetitive-dna-loci-and-their-modulation-by-the-non-canonical-nucleic-acid-structures-r-loops-and-g-quadruplexes
#6
Amanda C Hall, Lauren A Ostrowski, Violena Pietrobon, Karim Mekhail
Cells have evolved intricate mechanisms to maintain genome stability despite allowing mutational changes to drive evolutionary adaptation. Repetitive DNA sequences, which represent the bulk of most genomes, are a major threat to genome stability often driving chromosome rearrangements and disease. The major source of repetitive DNA sequences and thus the most vulnerable constituents of the genome are the rDNA (rDNA) repeats, telomeres, and transposable elements. Maintaining the stability of these loci is critical to overall cellular fitness and lifespan...
March 4, 2017: Nucleus
https://www.readbyqxmd.com/read/28375731/crispr-cas9-structures-and-mechanisms
#7
Fuguo Jiang, Jennifer A Doudna
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes...
March 30, 2017: Annual Review of Biophysics
https://www.readbyqxmd.com/read/28349431/detection-and-characterization-of-r-loop-structures
#8
Raquel Boque-Sastre, Marta Soler, Sonia Guil
R loops are special three stranded nucleic acid structures that comprise a nascent RNA hybridized with the DNA template strand, leaving a non-template DNA single-stranded. More specifically, R loops form in vivo as G-rich RNA transcripts invade the DNA duplex and anneal to the template strand to generate an RNA:DNA hybrid, leaving the non-template, G-rich DNA strand in a largely single-stranded conformation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012).DNA-RNA hybrids are a natural occurrence within eukaryotic cells, with levels of these hybrids increasing at sites with high transcriptional activity, such as during transcription initiation, repression, and elongation...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28349430/rna-fish-to-study-regulatory-rna-at-the-site-of-transcription
#9
Marta Soler, Raquel Boque-Sastre, Sonia Guil
The increasing role of all types of regulatory RNAs in the orchestration of cellular programs has enhanced the development of a variety of techniques that allow its precise detection, quantification, and functional scrutiny. Recent advances in imaging and fluoresecent in situ hybridization (FISH) methods have enabled the utilization of user-friendly protocols that provide highly sensitive and accurate detection of ribonucleic acid molecules at both the single cell and subcellular levels. We herein describe the approach originally developed by Stellaris(®), in which the target RNA molecule is fluoresecently labeled with multiple tiled complementary probes each carrying a fluorophore, thus improving sensitivity and reducing the chance of false positives...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28341774/rna-dna-hybrid-r-loop-immunoprecipitation-mapping-an-analytical-workflow-to-evaluate-inherent-biases
#10
László Halász, Zsolt Karányi, Beáta Boros-Oláh, Tímea Rózsa, Éva Sipos, Éva Nagy, Ágnes Mosolygó-L, Anett Mázló, Éva Rajnavölgyi, Gábor Halmos, Lóránt Székvölgyi
The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping...
March 24, 2017: Genome Research
https://www.readbyqxmd.com/read/28318821/snf2-family-protein-fft3-suppresses-nucleosome-turnover-to-promote-epigenetic-inheritance-and-proper-replication
#11
Nitika Taneja, Martin Zofall, Vanivilasini Balachandran, Gobi Thillainadesan, Tomoyasu Sugiyama, David Wheeler, Ming Zhou, Shiv I S Grewal
Heterochromatin can be epigenetically inherited in cis, leading to stable gene silencing. However, the mechanisms underlying heterochromatin inheritance remain unclear. Here, we identify Fft3, a fission yeast homolog of the mammalian SMARCAD1 SNF2 chromatin remodeler, as a factor uniquely required for heterochromatin inheritance, rather than for de novo assembly. Importantly, we find that Fft3 suppresses turnover of histones at heterochromatic loci to facilitate epigenetic transmission of heterochromatin in cycling cells...
April 6, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28314779/an-atr-dependent-function-for-the-ddx19-rna-helicase-in-nuclear-r-loop-metabolism
#12
Dana Hodroj, Bénédicte Recolin, Kamar Serhal, Susan Martinez, Nikolay Tsanov, Raghida Abou Merhi, Domenico Maiorano
Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops...
March 17, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28273945/crispr-cas9-cleavage-efficiency-correlates-strongly-with-target-sgrna-folding-stability-from-physical-mechanism-to-off-target-assessment
#13
Xiaojun Xu, Dongsheng Duan, Shi-Jie Chen
The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not fully understood. In this study, based on a statistical mechanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong correlation between Cas9 cleavage efficiency and the stability of the DNA-RNA (R-loop) complex structures, with a Pearson correlation coefficient ranging from 0...
December 2017: Scientific Reports
https://www.readbyqxmd.com/read/28270613/smn-deficiency-in-severe-models-of-spinal-muscular-atrophy-causes-widespread-intron-retention-and-dna-damage
#14
Mohini Jangi, Christina Fleet, Patrick Cullen, Shipra V Gupta, Shila Mekhoubad, Eric Chiao, Norm Allaire, C Frank Bennett, Frank Rigo, Adrian R Krainer, Jessica A Hurt, John P Carulli, John F Staropoli
Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear...
March 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28270450/ssb1-and-ssb2-cooperate-to-regulate-mouse-hematopoietic-stem-and-progenitor-cells-by-resolving-replicative-stress
#15
Wei Shi, Therese Vu, Didier Boucher, Anna Biernacka, Jules Nde, Raj K Pandita, Jasmin Straube, Glen M Boyle, Fares Al-Ejeh, Purba Nag, Jessie Jeffery, Janelle L Harris, Amanda L Bain, Marta Grzelak, Magdalena Skrzypczak, Abhishek Mitra, Norbert Dojer, Nicola Crosetto, Nicole Cloonan, Olivier J Becherel, John Finnie, Jeffrey R Skaar, Carl R Walkley, Tej K Pandita, Maga Rowicka, Krzysztof Ginalski, Steven W Lane, Kum Kum Khanna
Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The two single-stranded DNA binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however their overlapping roles during normal physiology are incompletely understood. We generated mice where both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, while conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double strand breaks (DSBs) enriched at Ssb binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA...
March 7, 2017: Blood
https://www.readbyqxmd.com/read/28268090/both-r-loop-removal-and-ribonucleotide-excision-repair-activities-of-rnase-h2-contribute-substantially-to-chromosome-stability
#16
Deborah A Cornelio, Hailey N C Sedam, Jessica A Ferrarezi, Nadia M V Sampaio, Juan Lucas Argueso
Cells carrying deletions of genes encoding H-class ribonucleases display elevated rates of chromosome instability. The role of these enzymes is to remove RNA-DNA associations including persistent mRNA-DNA hybrids (R-loops) formed during transcription, and ribonucleotides incorporated into DNA during replication. RNases H1 and H2 can degrade the RNA component of R-loops, but only RNase H2 can initiate accurate ribonucleotide excision repair (RER). In order to examine the specific contributions of these activities to chromosome stability, we measured rates of loss-of-heterozygosity (LOH) in diploid Saccharomyces cerevisiae yeast strains carrying the rnh201-RED separation-of-function allele, encoding a version of RNase H2 that is RER-defective, but partly retains its other activity...
April 2017: DNA Repair
https://www.readbyqxmd.com/read/28257700/functions-of-replication-protein-a-as-a-sensor-of-r-loops-and-a-regulator-of-rnaseh1
#17
Hai Dang Nguyen, Tribhuwan Yadav, Sumanprava Giri, Borja Saez, Timothy A Graubert, Lee Zou
R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops...
March 2, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28203566/increased-spontaneous-recombination-in-rnase-h2-deficient-cells-arises-from-multiple-contiguous-rnmps-and-not-from-single-rnmp-residues-incorporated-by-dna-polymerase-epsilon
#18
Anastasiya Epshtein, Catherine J Potenski, Hannah L Klein
Ribonucleotides can become embedded in DNA from insertion by DNA polymerases, failure to remove Okazaki fragment primers, R-loops that can prime replication, and RNA/cDNA-mediated recombination. RNA:DNA hybrids are removed by RNase H enzymes. Single rNMPs in DNA are removed by RNase H2 and if they remain on the leading strand, can lead to mutagenesis in a Top1-dependent pathway. rNMPs in DNA can also stimulate genome instability, among which are homologous recombination gene conversion events. We previously found that, similar to the rNMP-stimulated mutagenesis, rNMP-stimulated recombination was also Top1-dependent...
June 2016: Microbial Cell
https://www.readbyqxmd.com/read/28098815/replication-fork-protection-factors-controlling-r-loop-bypass-and-suppression
#19
REVIEW
Emily Yun-Chia Chang, Peter C Stirling
Replication-transcription conflicts have been a well-studied source of genome instability for many years and have frequently been linked to defects in RNA processing. However, recent characterization of replication fork-associated proteins has revealed that defects in fork protection can directly or indirectly stabilize R-loop structures in the genome and promote transcription-replication conflicts that lead to genome instability. Defects in essential DNA replication-associated activities like topoisomerase, or the minichromosome maintenance (MCM) helicase complex, as well as fork-associated protection factors like the Fanconi anemia pathway, both appear to mitigate transcription-replication conflicts...
January 14, 2017: Genes
https://www.readbyqxmd.com/read/28076779/transcription-dynamics-prevent-rna-mediated-genomic-instability-through-srpk2-dependent-ddx23-phosphorylation
#20
Sreerama Chaitanya Sridhara, Sílvia Carvalho, Ana Rita Grosso, Lina Marcela Gallego-Paez, Maria Carmo-Fonseca, Sérgio Fernandes de Almeida
Genomic instability is frequently caused by nucleic acid structures termed R-loops that are formed during transcription. Despite their harmful potential, mechanisms that sense, signal, and suppress these structures remain elusive. Here, we report that oscillations in transcription dynamics are a major sensor of R-loops. We show that pausing of RNA polymerase II (RNA Pol II) initiates a signaling cascade whereby the serine/arginine protein kinase 2 (SRPK2) phosphorylates the DDX23 helicase, culminating in the suppression of R-loops...
January 10, 2017: Cell Reports
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