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

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE200950 | GEO | 2022/04/23

REPOSITORIES: GEO

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

Project description: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.

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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

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

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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.

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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

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Encyclopedia of C. elegans 3' UTRs and their regulatory elements - 454 sequencing of RACE clones

2011-01-21 | E-GEOD-26795 | biostudies-arrayexpress

Encyclopedia of C. elegans 3' UTRs and their regulatory elements

Project description:The 3' untranslated region (3'UTR) constitutes a major site of post-transcriptional regulation of gene expression. Sequence elements in the 3'UTR interact with trans-acting regulators such as microRNAs that affect translation and stability. The overall aim is to use a 3'RACE cloning-sequencing stragety to identify the 3'UTRs of C. elegans transcripts and explore their heterogeneity in different developmental stages and tissues. Keywords: Transcriptome analysis For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Raw data files are available on our FTP site: ftp://ftp.ncbi.nlm.nih.gov/pub/geosup/Series/GSE17781

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Encyclopedia of C. elegans 3' UTRs and their regulatory elements

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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.

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