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QuantSeq/3' targeted seq supporting study Probing 3’UTRs as modular regulators of gene expression

ABSTRACT: The goal of the study is to analyze the function of 3'-UTR motifs in the non-native context. Several 3'-UTR motifs predicted to have stabilizing or destabilizing effects on mRNA were inserted into terminators controlling the expression of reporter mCherry gene in following contexts: pRps3-mCherry-tRps3, pPgk1-mCherry-tRps3, pTsa1-mCherry-tTsa. 3'-end sequencing was used to measure the levels of expression of reporter gene and distribution of poly(A) sites.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE200211 | GEO | 2022/04/09

REPOSITORIES: GEO

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5PSeq supporting study Probing 3’UTRs as modular regulators of gene expression

Project description:The goal of the study is to analyse the function of 3'-UTR motifs in the non-native context. Several 3'-UTR motifs predicted to have stabilizing or destabilizing effects on mRNA were inserted into terminators controlling the expression of reporter mCherry gene in following contexts: pRps3-mCherry-tRps3, pPgk1-mCherry-tRps3, pTsa1-mCherry-tTsa. 5'P sequencing was used to determine whether or not modifications in the 3'-UTR region affect ribosomal dynamics and co-translational degradation of mRNA in the 3' end region of reporter coding sequence.

2022-04-23 | GSE200950 | GEO

Cas13d experimental viral genome-wide screen identifies regions of RNA vulnerability in SARScov2

Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.

2023-10-29 | E-MTAB-11111 | biostudies-arrayexpress

QuantSeq/3' targeted seq supporting study Probing 3’UTRs as modular regulators of gene expression

Project description:QuantSeq/3' targeted seq supporting study Probing 3’UTRs as modular regulators of gene expression

| PRJNA823690 | ENA

5PSeq supporting study Probing 3’UTRs as modular regulators of gene expression

Project description:5PSeq supporting study Probing 3’UTRs as modular regulators of gene expression

| PRJNA827707 | ENA

Targeted DMS probing of SF3A3 5' UTR

Project description:To delineate the native structure of SF3A3 5'UTR, RNA was harvested from IMR90 human fibroblasts. Using specific primers and DMS-MaPSeq pipeline, we validated individual base pairing probabilities within the endogenous 5'UTR of SF3A3 (samples described as 'in vivo' transcribed). DMS-MaP-Seq is based on the principle that DMS is highly reactive to solvent-accessible, unpaired adenine (A) and cytosine (C) residues, but remains inert toward base-paired A and C engaged in Watson-Crick interactions (Rouskin et al., 2014). Using this methodology, we identify stable stem-loop structure (SL3) positioned within SF3A3 5'UTR. To further validate the functional importance of SL3, the structural point mutant (SF3A3 5'UTR mut: A55C and U95A) and rescue (SF3A3 5'UTR res: A55C and U95A and rescuing point mutations G61U and U100G) sequences of SF3A3 5'UTR were cloned into the reporter plasmid. For the validation of these mutate-and-rescue constructs, plasmids were in vitro transcribed and either used directly (samples described as 'in vitro') for DMS-MaP-Seq probing.

2021-03-25 | GSE147504 | GEO

mRNA-seq analysis of Caenorhabditis elegans daf-18(ok480) and WT starved L1 larvae shortly after hatching

Project description:Whole L1 larvae were collected from GC1459 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III ?; daf-18(ok480) IV] and GC1171 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III] strains up to two hours after hatching without food.

2021-06-21 | GSE151299 | GEO

Gene expression analysis of hESC-derived-MESP1 expressing progenitors

Project description:A hESC MESP1-MCHERRY reporter line was used to isolate and study the molecular character of MESP1 expressing pre-cardiac progenitors, derived from hESC. MESP1 is a key-transcription factor for pre-cardiac mesoderm and is marking the progenitor for almost all cells of the heart. This reporter line was used to study cardiac differentiation and the derivation of early cardiac progenitors in vitro. hESCs were differentiated towards the cardiac lineage, expressing MESP1-mCherry at day 3 of differentiation. Total RNA obtained from isolated MESP1-mCherry expressing progenitors was compared to that of non-MESP1-expressing progenitors and undifferentiated hESCs in order to characterize MESP1-specific transcription factors and proteins.

2014-09-01 | E-GEOD-56721 | biostudies-arrayexpress

Denaturing CLIP (dCLIP) pipeline identifies discrete RNA footprints on chromatin-associated proteins and reveals that CBX7 targets 3’UTRs to regulate mRNA expression

Project description:Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP), and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA-binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3’ untranslated regions (UTRs) of messenger RNAs. CBX7 binds with a median RNA “footprint” of 171-183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These data support the utility of dCLIP and reveal an unexpected functional interaction between CBX7 and the 3’UTRs of mRNA.interacts with a range of transcripts, it predominantly binds 3’ untranslated regions (UTRs) of messenger RNAs. CBX7 has a median RNA “footprint” of 171 nucleotides, enabling identification of four families of motifs that tend to co-cluster in the 3’UTR. Manipulating the CBX7-3’UTR interactions and motifs with antisense oligonucleotides results in upregulation of mRNA and protein expression, thereby validating the dCLIP method and revealing functional interactions between CBX7 and mRNA.

2017-10-25 | GSE88823 | GEO

scRNAseq of Cyp11a1-expressing cells from mouse melanoma model derived tumors

Project description:Tumours induce de novo steroidogenesis in immune cells. We wanted to define the gene expression identity of intratumoural steroidogenic immune cells and compare them with the transcriptome with non-steroidogenic cells. Cyp11a1 expression is a faithful biomarker of de novo steroidogenesis (i.e. Cyp11a1+ cells are steroidogenic and Cyp11a1- cells are non-steroidogenic). We inoculated B16-F10 tumor in Cyp11a1-mCherry reporter mice, enriched and purified intratumoral Cyp11a1-mCherry+ and Cyp11a1-mCherry- cells by cell sorting into 96-well plates [with a ratio of 79:15 (mCherry+ : mCherry-) cells per plate] and performed scRNA-seq using SMART-Seq2 platform.

2020-05-27 | E-MTAB-8509 | biostudies-arrayexpress

A Human Embryonic Stem Cell Reporter Line for Studying Cardiomyocyte Biology

Project description:Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes for studying cardiac biology, disease modeling, drug screens, and potentially for regenerative therapies. Directed differentiation protocols for cardiomyocytes using hESCs are well established, but methods to isolate highly pure population of cardiomyocytes are limited. Reporter cell lines can be valuable for purification and visualization of cells for such applications. We used CRISPR/Cas9 in hESCs to place an mCherry reporter gene into the MYH6 locus, facilitating a simple method to purify cardiomyocytes. MYH6:mCherry positive cells express atrial and ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human cardiomyocytes and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiomyocyte arrhythmia. The MYH6:mCherry hESC reporter line should serve as a useful tool for disease modeling and drug development relevant to cardiomyocyte biology.

2019-01-01 | GSE111365 | GEO

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