RNA Modification & Editing

The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing

Thu, 01/08/2015 - 00:00
Alon S, Garrett SC, Levanon EY, Olson S, Graveley BR, Rosenthal JJ, Eisenberg E.
eLIFE
Eric Boyer
Time
12:00pm
RNA editing by adenosine deamination alters genetic information from the genomic blueprint. When it recodes mRNAs, it gives organisms the option to express diverse, functionally distinct, protein isoforms. All eumetazoans, from cnidarians to humans, express RNA editing enzymes. However, transcriptome-wide screens have only uncovered about 25 transcripts harboring conserved recoding RNA editing sites in mammals and several hundred recoding sites in Drosophila. These studies on few established models have led to the general assumption that recoding by RNA editing is extremely rare. Here we employ a novel bioinformatic approach with extensive validation to show that the squid Doryteuthis pealeii recodes proteins by RNA editing to an unprecedented extent. We identify 57,108 recoding sites in the nervous system, affecting the majority of the proteins studied. Recoding is tissue-dependent, and enriched in genes with neuronal and cytoskeletal functions, suggesting it plays an important role in brain physiology
RNA Modification & Editing
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eboyer
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RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

Tue, 08/05/2014 - 00:00
Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L, Li F, Luo B, Alvarez-Carbonell D, Garcia-Mesa Y, Karn J, Mo X, Khalili K.
Proc Natl Acad Sci U S A
Daniele Cary
Time
12:00pm
AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor offtarget editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9- expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.
RNA Modification & Editing
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carydc
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Saturation editing of genomic regions by multiplex homology-directed repair

Thu, 09/04/2014 - 00:00
Findlay GM, Boyle EA, Hause RJ, Klein JC, Shendure J.
Nature
Jacob Freimer
Time
12:00pm
Saturation mutagenesis1,2—coupled to an appropriate biological assay—represents a fundamentalmeans of achieving a high-resolution understanding of regulatory3 and protein-coding4 nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or ‘‘safe-harbour’’ integration fail to capture the native context of the endogenous chromosomal locus5 . This shortcoming markedly limits the interpretability of the resultingmeasurements ofmutationalimpact.Here,we coupleCRISPR/ Cas9 RNA-guided cleavage6withmultiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a sixbase-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsensemediated decay and exonic splicing elements.We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences oflarge numbers of mutationswith saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.
 
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Translating dosage compensation to trisomy 21

Thu, 08/15/2013 - 00:00
Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM, Carone DM, Carone BR, Shivak DA, Guschin DY, Pearl JR, Rebar EJ, Byron M, Gregory PD, Brown CJ, Urnov FD, Hall LL, Lawrence JB.
Nature
Juan Moreno Moya
Time
12:00pm
Down syndrome (DS) is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21 (Chr21). We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST. Using genome editing with zinc finger nucleases, we targeted a large, inducible XIST transgene into the Chr21 DYRK1A locus, in DS pluripotent stem cells. XIST RNA coats Chr21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a “Chr21 Barr Body.” This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Remarkably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one Chr21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of “chromosome therapy”.
 
RNA Modification & Editing
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jmmormoy
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Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

Thu, 01/17/2013 - 00:00
Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR
Nature
Julia Ye
Time
12:00pm
A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes1 . Similar to RNA interference in eukaryotes2 , these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes3 . Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of antiCRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.
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RNA Editing Underlies Temperature Adaptation in K+ Channels from Polar Octopi

Fri, 02/17/2012 - 00:00
Sandra Garrett, Joshua J.C. Rosenthal
Science
Malika Morar
Time
12:00pm
To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here, we show that delayed rectifier K+ channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very similar behavior when expressed in Xenopus oocytes. However, the transcribed messenger RNAs are extensively edited, creating functional diversity. One editing site, which recodes an isoleucine to a valine in the channel’s pore, greatly accelerates gating kinetics by destabilizing the open state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus adenosine-to-inosine RNA editing can respond to the physical environment.
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Germline deletion of the miR-17∼92 cluster causes skeletal and growth defects in humans.

Thu, 10/20/2011 - 00:00
de Pontual L, Yao E, Callier P, Faivre L, Drouin V, Cariou S, Van Haeringen A, Geneviève D, Goldenberg A, Oufadem M, Manouvrier S, Munnich A, Vidigal JA, Vekemans M, Lyonnet S, Henrion-Caude A, Ventura A, Amiel J
Nat Genet. 2011 Sep 4;43(10):1026-30. doi: 10.1038/ng.915.
Malika Morar
Time
12:00pm
MicroRNAs (miRNAs) are key regulators of gene expression in animals and plants. Studies in a variety of model organisms show that miRNAs modulate developmental processes. To our knowledge, the only hereditary condition known to be caused by a miRNA is a form of adult-onset non-syndromic deafness1, and no miRNA mutation has yet been found to be responsible for any developmental defect in humans. Here we report the identification of germline hemizygous deletions of MIR17HG, encoding the miR-17~92 polycistronic miRNA cluster, in individuals with microcephaly, short stature and digital abnormalities. We demonstrate that haploinsufficiency of miR-17~92 is responsible for these developmental abnormalities by showing that mice harboring targeted deletion of the miR-17~92 cluster phenocopy several key features of the affected humans. These findings identify a regulatory function for miR-17~92 in growth and skeletal development and represent the first example of an miRNA gene responsible for a syndromic developmental defect in humans.
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CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III

Thu, 03/31/2011 - 00:00
Elitza Deltcheva, Krzysztof Chylinski, Cynthia M. Sharma, Karine Gonzales, Yanjie Chao, Zaid A. Pirzada, Maria R. Eckert, Jörg Vogel, Emmanuelle Charpentier
Nature
Michael McManus
Time
12:00pm
CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.
RNA Modification & Editing
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Target RNA-directed trimming and tailing of small silencing RNAs

Fri, 06/18/2010 - 00:00
Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z, Zamore PD.
Science
Mark Ansel
Time
12:00pm
In Drosophila, microRNAs (miRNAs) typically guide Argonaute1 to repress messenger RNA (mRNA), whereas small interfering RNAs (siRNAs) guide Argonaute2 to destroy viral and transposon RNA. Unlike siRNAs, miRNAs rarely form extensive numbers of base pairs to the mRNAs they regulate. We find that extensive complementarity between a target RNA and an Argonaute1-bound miRNA triggers miRNA tailing and 3′-to-5′ trimming. In flies, Argonaute2-bound small RNAs—but not those bound to Argonaute1—bear a 2′-O-methyl group at their 3′ ends. This modification blocks target-directed small RNA remodeling: In flies lacking Hen1, the enzyme that adds the 2′-O-methyl group, Argonaute2-associated siRNAs are tailed and trimmed. Target complementarity also affects small RNA stability in human cells. These results provide an explanation for the partial complementarity between animal miRNAs and their targets.
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