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Series GSE179744 Query DataSets for GSE179744
Status Public on May 04, 2023
Title Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites
Organism Mus musculus
Experiment type Other
Summary Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (Nuclear RNAi defective-2) and CCDC174 (Coiled-Coil Domain-Containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and unspliced 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use non-canonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice and accurate pre-mRNA splicing.
 
Overall design All BCLIP-seq, RNA-seq and Ribo-seq experiments were performed in two independent replicates for each sample. BCLIP-seq was done with proteins endogenously tagged with a composite 3xFLAG+AviTag (3A tag). Nrde2 and Mtrex knockout were induced by treating the conditional knockout mES cells with 100 nM 4-hydroxytamoxifen for 4 and 3 days, respectively. Depletion of all the proteins fused to FKBP12F36V degron was achieved by treating the cells with 500 nM dTAG-13 compound for 24 hours. The indicated RNA-seq samples were treated for 4 hours with 0.1 mg/ml cycloheximide (CHX).
 
Contributor(s) Flemr M, Schwaiger M, Mohn F, Bühler M
Citation(s) 37137667
Submission date Jul 08, 2021
Last update date May 09, 2023
Contact name Fabio Mohn
E-mail(s) fabio.mohn@fmi.ch
Organization name Friedrich Miescher Institute for Biomedical Research
Lab Buehler Lab
Street address Maulbeerstrasse 66
City Basel
ZIP/Postal code 4058
Country Switzerland
 
Platforms (1)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
Samples (48)
GSM5430372 BCLIPseq of NRDE2 in wt mES cells, replicate 1
GSM5430373 BCLIPseq of NRDE2 in wt mES cells, replicate 2
GSM5430374 BCLIPseq of NRDE2d200 in mES cells, replicate 1
Relations
BioProject PRJNA744888
SRA SRP327516

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE179744_RAW.tar 4.1 Gb (http)(custom) TAR (of BIGWIG, BW)
GSE179744_RNAseq_cpm_per_gene.txt.gz 3.5 Mb (ftp)(http) TXT
GSE179744_Riboseq_cpm_per_gene.txt.gz 481.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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