scholarly journals Analysis of Human Small Nucleolar RNAs (snoRNA) and the Development of snoRNA Modulator of Gene Expression Vectors

2010 ◽  
Vol 21 (9) ◽  
pp. 1569-1584 ◽  
Author(s):  
Motoharu Ono ◽  
Kayo Yamada ◽  
Fabio Avolio ◽  
Michelle S. Scott ◽  
Silvana van Koningsbruggen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Cellular Protein ◽  
Vector System ◽  
Expression Vectors ◽  
Small Nucleolar Rnas ◽  
Rna Targets ◽  
Internal Sequence ◽  
Single Transcript ◽  
Nucleolar Rnas

Human small nucleolar RNAs (snoRNAs) that copurify with nucleoli isolated from HeLa cells have been characterized. Novel fibrillarin-associated snoRNAs were detected that allowed the creation of a new vector system for the targeted knockdown of one or more genes in mammalian cells. The snoMEN (snoRNA modulator of gene expressioN) vector technology is based on snoRNA HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets. Gene-specific knockdowns are demonstrated for endogenous cellular proteins and for G/YFP-fusion proteins. Multiplex snoMEN vectors coexpress multiple snoRNAs in one transcript, targeted either to different genes or to different sites in the same gene. Protein replacement snoMEN vectors can express a single transcript combining cDNA for a tagged protein with introns containing cognate snoRNAs targeted to knockdown the endogenous cellular protein. We foresee applications for snoMEN vectors in basic gene expression research, target validation, and gene therapy.

scholarly journals Construction, Rescue, and Characterization of Vectors Derived from Ovine Atadenovirus

2003 ◽  
Vol 77 (22) ◽  
pp. 11941-11951 ◽  
Author(s):  
Peter Löser ◽  
Christian Hofmann ◽  
Gerald W. Both ◽  
Wolfgang Uckert ◽  
Moritz Hillgenberg
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Human Factor ◽  
Insertion Site ◽  
Factor Ix ◽  
Vector System ◽  
Virus Rescue ◽  
Human Factor Ix

ABSTRACT Gene transfer vectors derived from ovine atadenovirus type 7 (OAdV) can efficiently infect a variety of mammalian cells in vitro and in vivo to deliver and express transgenes. However, early OAdV vectors were designed on human mastadenovirus principles prior to the complete characterization of OAdV genes and transcripts. The distinctive arrangement of the OAdV genome has suggested ways to improve OAdV vector design and utility. We therefore developed a cosmid-based approach that allows efficient construction of recombinant ovine atadenovirus genomes in which the transgene is inserted at one of three sites. Viruses were rescued by transfection of viral DNA into a new ovine fetal skin fibroblast producer cell line, HVO156. The suitability of the three insertion sites was compared with respect to virus rescue efficiency, gene expression levels, and genetic stability of the vectors. We found that one vector with a transgene inserted at site 1, between the pVIII and fiber genes, was unstable. Only one vector that carried a transgene at site 2, near the right end of the genome, together with a nearby deletion was rescued. In contrast, several vectors with different transgenes inserted in site 3, between the E4 and RH transcription units, were repeatedly rescued, and these vectors were stable over at least four passages. Transgene orientation in site 3 had only little effect on expression. Finally, a vector carrying a human factor IX cDNA at site 3, when administered intravenously, produced nearly physiological levels of human factor IX in mice. The availability of an efficient method for vector construction and the identification of a new insertion site for virus rescue and gene expression substantially enhance the utility of the OAdV vector system.

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2439-2449 ◽  
Author(s):  
Elisabeth Sørvig ◽  
Geir Mathiesen ◽  
Kristine Naterstad ◽  
Vincent G. H. Eijsink ◽  
Lars Axelsson
Keyword(s):  
Gene Expression ◽  
Lactobacillus Plantarum ◽  
Modular Design ◽  
Regulatory Elements ◽  
Cellular Protein ◽  
Expression Vectors ◽  
Lactobacillus Sakei ◽  
Inducible Gene Expression ◽  
Set Up ◽  
Inducible Gene

Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called ‘pSIP’ vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using β-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein.

scholarly journals Mapping targets for small nucleolar RNAs in yeast

2018 ◽  
Vol 3 ◽  
pp. 120
Author(s):  
Tatiana Dudnakova ◽  
Hywel Dunn-Davies ◽  
Rosie Peters ◽  
David Tollervey
Keyword(s):  
Binding Sites ◽  
High Energy ◽  
Small Nucleolar Rnas ◽  
High Confidence ◽  
Regulatory Interactions ◽  
Uv Crosslinking ◽  
Rna Targets ◽  
Bona Fide ◽  
Target Binding ◽  
Nucleolar Rnas

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions.

Nucleolar RNPs: from genes to functional snoRNAs in plants

2010 ◽  
Vol 38 (2) ◽  
pp. 672-676 ◽  
Author(s):  
Julie Rodor ◽  
Ingrid Letelier ◽  
Loreto Holuigue ◽  
Manuel Echeverria
Keyword(s):  
Protein Interactions ◽  
Small Rnas ◽  
Small Nucleolar Rnas ◽  
Protein Protein Interactions ◽  
Nucleolar Proteins ◽  
Rna Targets ◽  
Complex Process ◽  
Small Nuclear Rnas ◽  
Nucleolar Rnas

The snoRNAs (small nucleolar RNAs) and related scaRNAs (small RNAs in the Cajal bodies) represent a major class of nuclear RNAs that guide 2′-O-ribose methylation and pseudouridylation of rRNAs, snRNAs (small nuclear RNAs) and other RNA targets. In vivo, all snoRNAs associate with a set of four highly conserved nucleolar proteins, forming the functional snoRNPs (small nucleolar ribonucleoproteins). The core structure of these mature snoRNPs has now been well described in eukaryotes, but less is known of their biogenesis. Recent data in animals and yeast reveal that assembly of the snoRNPs is a complex process that implicates several auxiliary proteins and transient protein–protein interactions. This new level of snoRNP regulation is now beginning to be unravelled in animals and yeast, but remains unexplored in plants. In the present paper, we review recent data from genomic and functional analysis allowing the identification and study of factors controlling the biogenesis of plant snoRNPs and their impact on plant development.

scholarly journals Controlling gene expression in mammalian cells using multiplexed conditional guide RNAs for Cas12a

2021 ◽  
Author(s):  
Lukas Oesinghaus ◽  
Friedrich C. Simmel
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Basic Research ◽  
Dependent Manner ◽  
Multiple Control ◽  
Guide Rnas ◽  
Single Transcript ◽  
Spatiotemporal Control ◽  
Expression In Mammalian Cells ◽  
Activate Gene

AbstractSpatiotemporal control of the activity of Cas proteins is of considerable interest for both basic research and therapeutics. Only few mechanisms have been demonstrated for regulating the activity of guide RNAs (gRNAs) for Cas12a in mammalian cells, however, and combining and compactly integrating multiple control instances on single transcripts has not been possible so far. Here, we show that conditional processing of the 3’ tail is a viable general approach towards switchable Pol II-transcribed Cas12a gRNAs that can activate gene expression in mammalian cells in an input-dependent manner. Processing of the 3’ tail can be achieved using microRNA and short hairpin RNA as inputs, via a guanine-responsive ribozyme, and also using an RNA strand displacement mechanism. We further show that Cas12a along with several independently switchable gRNAs can be integrated on a single transcript using stabilizing RNA triplexes, providing a route towards compact Cas12a-based gene regulation constructs with multi-input switching capabilities.

scholarly journals Imaging Real-Time Gene Expression in Mammalian Cells with Single-Transcript Resolution: Figure 1.

2007 ◽  
Vol 2007 (11) ◽  
pp. pdb.prot4869 ◽  
Author(s):  
Amber L. Wells ◽  
John S. Condeelis ◽  
Robert H. Singer ◽  
Daniel Zenklusen
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Mammalian Cells ◽  
Single Transcript ◽  
Expression In Mammalian Cells

scholarly journals Mapping targets for small nucleolar RNAs in yeast

2018 ◽  
Vol 3 ◽  
pp. 120 ◽  
Author(s):  
Tatiana Dudnakova ◽  
Hywel Dunn-Davies ◽  
Rosie Peters ◽  
David Tollervey
Keyword(s):  
Binding Sites ◽  
High Energy ◽  
Small Nucleolar Rnas ◽  
High Confidence ◽  
Regulatory Interactions ◽  
Uv Crosslinking ◽  
Rna Targets ◽  
Bona Fide ◽  
Target Binding ◽  
Nucleolar Rnas

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions.

scholarly journals Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome

2015 ◽  
Vol 112 (52) ◽  
pp. 15916-15921 ◽  
Author(s):  
Sneha Rath ◽  
Jesse Donovan ◽  
Gena Whitney ◽  
Alisha Chitrakar ◽  
Wei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Innate Immune System ◽  
Mammalian Cells ◽  
Innate Immune ◽  
Rnase L ◽  
Double Stranded Rna ◽  
Rna Targets ◽  
Human Rnase ◽  
Cell Growth And Differentiation ◽  
Cell Adhesion And Proliferation

Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.

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