scholarly journals Identification and analysis of functional associations among natural eukaryotic genome editing components

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1374 ◽  
Author(s):  
Estienne C. Swart ◽  
Cyril Denby Wilkes ◽  
Pamela Y. Sandoval ◽  
Cristina Hoehener ◽  
Aditi Singh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Small Rnas ◽  
Rna Binding ◽  
Elongation Factor ◽  
Chromatin Remodelling ◽  
Eukaryotic Genome ◽  
Functional Relationships ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Main Components

During development in the ciliate Paramecium, excess DNA interspersed throughout the germline genome is deleted to generate a new somatic genome. In this process, most of the intervening DNA is excised by a Piggybac-derived transposase, assisted by small RNAs (scnRNAs and iesRNAs) and chromatin remodelling. As the list of genes involved in DNA elimination has been growing, a need for a general approach to discover functional relationships among these genes now exists. We show that deep sequencing-based comparisons of experimentally-induced DNA retention provide a sensitive, quantitative approach to identify and analyze functional associations among genes involved in native genome editing. This reveals two functional molecular groups: (i) iesRNAs/scnRNAs, the putative Piwi- and RNA-binding Nowa1/2 proteins, and the transcription elongation factor TFIIS4; and (ii) PtCAF1 and Ezl1, two proteins involved in chromatin remodelling. Comparative analyses of silencing effects upon the largely unstudied regions comprising most developmentally eliminated DNA in Paramecium suggests a continuum between precise and imprecise DNA elimination. These findings show there is now a way forward to systematically elucidate the main components of natural eukaryotic genome editing systems.

scholarly journals RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA.

2022 ◽  
Author(s):  
Aditi Singh ◽  
Xyrus X. Maurer-Alcalá ◽  
Therese Solberg ◽  
Silvan Gisler ◽  
Michael Ignarski ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Chromatin Remodeling ◽  
Dna Cleavage ◽  
Small Rnas ◽  
Heterochromatin Formation ◽  
Dna Accessibility ◽  
Dna Elimination ◽  
Nucleosome Density ◽  
Somatic Genome ◽  
Genomic Regions

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.

scholarly journals Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises

2021 ◽  
Vol 11 ◽  
Author(s):  
Mikel Irastortza-Olaziregi ◽  
Orna Amster-Choder
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Molecular Architecture ◽  
Independent Manner ◽  
New Information ◽  
Shed Light

Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming “RNAP.mRNA.ribosome” complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate.

scholarly journals Equine Adipose-Derived Mesenchymal Stromal Cells Release Extracellular Vesicles Enclosing Different Subsets of Small RNAs

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Stefano Capomaccio ◽  
Katia Cappelli ◽  
Cinzia Bazzucchi ◽  
Mauro Coletti ◽  
Rodolfo Gialletti ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Small Rnas ◽  
Rna Binding ◽  
Bioinformatic Analysis ◽  
Protein Coding ◽  
Significant Enrichment ◽  
Pathway Analyses

Background. Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role. Methods. EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was isolated, enriched for small RNAs (<200 nt), and sequenced by Illumina technology. After bioinformatic analysis with state-of-the-art pipelines for short sequences, mapped reads were used to describe EV RNA cargo, reporting classes, and abundances. Enrichment analyses were performed to infer involved pathways and functional categories. Results. Electron microscopy showed the presence of vesicles ranging in size from 30 to 300 nm and expressing typical markers. RNA analysis revealed that ribosomal RNA was the most abundant fraction, followed by small nucleolar RNAs (snoRNAs, 13.67%). Miscellaneous RNA (misc_RNA) reached 4.57% of the total where Y RNA, RNaseP, and vault RNA represented the main categories. miRNAs were sequenced at a lower level (3.51%) as well as protein-coding genes (1.33%). Pathway analyses on the protein-coding fraction revealed a significant enrichment for the “ribosome” pathway followed by “oxidative phosphorylation.” Gene Ontology analysis showed enrichment for terms like “extracellular exosome,” “organelle envelope,” “RNA binding,” and “small molecule metabolic process.” The miRNA target pathway analysis revealed the presence of “signaling pathways regulating pluripotency of stem cells” coherent with the source of the samples. Conclusion. We herein demonstrated that e-AdMSC release EVs enclosing different subsets of small RNAs that potentially regulate a number of biological processes. These findings shed light on the role of EVs in the context of MSC biology.

scholarly journals Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Matthew J Shurtleff ◽  
Morayma M Temoche-Diaz ◽  
Kate V Karfilis ◽  
Sayaka Ri ◽  
Randy Schekman
Keyword(s):  
Cell Migration ◽  
Membrane Proteins ◽  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Target Cells ◽  
Rna Packaging ◽  
Exosome Biogenesis ◽  
Purification Scheme ◽  
A Cell

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.

scholarly journals Rapid modelling of cooperating genetic events in cancer through somatic genome editing

Nature ◽  
2014 ◽  
Vol 516 (7531) ◽  
pp. 428-431 ◽  
Author(s):  
Francisco J. Sánchez-Rivera ◽  
Thales Papagiannakopoulos ◽  
Rodrigo Romero ◽  
Tuomas Tammela ◽  
Matthew R. Bauer ◽  
...  
Keyword(s):  
Genome Editing ◽  
Somatic Genome ◽  
Genetic Events

scholarly journals The Time Is Ripe for Somatic Genome Editing: NIH Program to Strengthen Translation

2018 ◽  
Vol 26 (3) ◽  
pp. 671-674 ◽  
Author(s):  
Boris Fehse ◽  
Ulrike Abramowski-Mock
Keyword(s):  
Genome Editing ◽  
Somatic Genome

scholarly journals Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

2015 ◽  
Vol 29 (14) ◽  
pp. 1576-1585 ◽  
Author(s):  
Shin-Heng Chiou ◽  
Ian P. Winters ◽  
Jing Wang ◽  
Santiago Naranjo ◽  
Crissy Dudgeon ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Genome Editing ◽  
Viral Vector ◽  
Cancer Modeling ◽  
Somatic Genome ◽  
Vector Delivery

scholarly journals Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for precision tumor modeling

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Barbara Oldrini ◽  
Álvaro Curiel-García ◽  
Carolina Marques ◽  
Veronica Matia ◽  
Özge Uluçkan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Somatic Genome ◽  
Tumor Modeling

scholarly journals LIA4Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

2014 ◽  
Vol 13 (10) ◽  
pp. 1300-1311 ◽  
Author(s):  
Scott A. Horrell ◽  
Douglas L. Chalker
Keyword(s):  
Critical Role ◽  
Dna Rearrangement ◽  
Rnai Machinery ◽  
Content Type ◽  
Heterochromatin Formation ◽  
H3k27 Methylation ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Subsequent Elimination ◽  
Elimination Pathway

ABSTRACTExtensive DNA elimination occurs as part of macronuclear differentiation duringTetrahymenasexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway.LIA4knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, withoutLIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus duringTetrahymenamacronuclear differentiation.

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