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Series GSE209857 Query DataSets for GSE209857
Status Public on Jul 28, 2022
Title RNA structure landscape of S. cerevisiae introns
Organism Saccharomyces cerevisiae
Experiment type Other
Summary Pre-mRNA secondary structures are hypothesized to play widespread roles in regulating RNA processing pathways, but these structures have been difficult to visualize in vivo. Here, we characterize S. cerevisiae pre-mRNA structures through transcriptome-wide dimethyl sulfate (DMS) probing, enriching for low-abundance pre-mRNA through splicing inhibition. These data enable evaluation of structures from phylogenetic and mutational studies as well as identification of new structures across 161 introns. We find widespread formation of “zipper stems” between the 5’ splice site and branch point, “downstream stems” between the branch point and the 3’ splice site, and previously uncharacterized long stems that distinguish pre-mRNA from spliced mRNA. Multi-dimensional chemical mapping reveals that intron structures can form in vitro without the presence of binding partners, and structure ensemble prediction suggests that these structures appear in introns across the Saccharomyces genus. We develop the functional assay VARS-seq to characterize variants of RNA structure in high-throughput and we apply the method on 135 sets of stems across 7 introns, finding that some structured elements can increase spliced mRNA levels despite being distal from canonical splice sites. Unexpectedly, other structures including zipper stems can increase retained intron levels. This transcriptome-wide inference of intron RNA structures suggests new ideas and model systems for understanding how pre-mRNA folding influences gene expression.
Overall design 1. RNA structure probing after splicing inhibition in yeast: RNA was treated with DMS and reverse transcription with TGIRT (DMS-MaPseq) for samples and controls. The samples include: two replicates of DMS treatment after splicing inhibition (one replicate with 3 lanes of sequencing, and another with one lane), one sample for DMS treatment without splicing inhibition, and one sample for a no-modification control with splicing inhibition. 2. Splicing measurements for intron variant library: Intron structure variant libraries were integrated into the S. cerevisiae genome in their native gene context. Splicing levels were measured with RNA-sequencing, and gDNA sequencing was used to identify intron variants by pairing randomized barcodes to intron sequences.
Contributor(s) Rangan R, Huang R, Hunter O, Pham P, Ares M Jr, Das R
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Submission date Jul 27, 2022
Last update date Jun 24, 2023
Contact name Rhiju Das
Organization name Stanford University
Street address 279 Campus Dr, B419 Beckman Center
State/province CA
ZIP/Postal code 94305
Country USA
Platforms (3)
GPL17143 Illumina MiSeq (Saccharomyces cerevisiae)
GPL21656 Illumina HiSeq 4000 (Saccharomyces cerevisiae)
GPL27812 Illumina NovaSeq 6000 (Saccharomyces cerevisiae)
Samples (6)
GSM6402544 DMS_pladB_Rep1
GSM6402545 DMS_pladB_Rep2
GSM6402546 DMS_no_pladB
BioProject PRJNA862719

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Supplementary file Size Download File type/resource
GSE209857_RAW.tar 15.4 Mb (http)(custom) TAR (of TAR)
GSE209857_d1_d2_combined_rfcount_rfnorm_processed.tar.gz 674.9 Kb (ftp)(http) TAR
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Raw data are available in SRA
Processed data are available on Series record
Processed data provided as supplementary file

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