Refer to Data Download for additional information.
Public data: https://www.ebi.ac.uk/ena/browser/view/PRJNA961731
Manuscript:
Son S, Kim B, Yang J, Kim VN. Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing. Genes Dev. 2023 May 1;37(9-10):383-397.
doi: 10.1101/gad.350275.122. Epub 2023 May 26. PMID: 37236670; PMCID: PMC10270192. https://genesdev.cshlp.org/content/37/9-10/383.long
Background:
DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well-documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins within introns. We identified a DROSHA isoform (p140) lacking the PRD produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD’s enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.
Overall design:
Comparative miRNA expression profiling analysis of small RNA-seq data for:
(1) full-length DROSHA or DROSHA-p140 rescue in HCT116 or HEK293E DROSHA knockout (KO) cells and
(2) HCT116 WT cells cultured with media supplemented with 9.1 % or 0.1 % FBS.
Sample metadata:
sample | group | treatment | cell | replicate |
SRR24302008 | WT | 0.1 % FBS, 72 hr | HCT116 | rep3 |
SRR24302009 | WT | 0.1 % FBS, 72 hr | HCT116 | rep2 |
SRR24302010 | WT | 0.1 % FBS, 72 hr | HCT116 | rep1 |
SRR24302011 | WT | 9.1 % FBS, 72 hr | HCT116 | rep3 |
SRR24302012 | WT | 9.1 % FBS, 72 hr | HCT116 | rep2 |
SRR24302013 | WT | 9.1 % FBS, 72 hr | HCT116 | rep1 |
SRR24302014 | DROSHA_KO | GFP | HCT116 | rep1 |
SRR24302015 | WT | no treatment | HCT116 | rep1 |
SRR24302016 | DROSHA_KO | DROSHA-p140 | HEK293E | rep3 |
SRR24302017 | DROSHA_KO | DROSHA-p140 | HEK293E | rep2 |
SRR24302018 | DROSHA_KO | DROSHA-p140 | HEK293E | rep1 |
SRR24302019 | DROSHA_KO | full-length DROSHA | HEK293E | rep3 |
SRR24302020 | DROSHA_KO | full-length DROSHA | HEK293E | rep2 |
SRR24302021 | DROSHA_KO | full-length DROSHA | HEK293E | rep1 |
SRR24302022 | DROSHA_KO | DROSHA-p140 | HCT116 | rep3 |
SRR24302023 | DROSHA_KO | DROSHA-p140 | HCT116 | rep2 |
SRR24302024 | DROSHA_KO | DROSHA-p140 | HCT116 | rep1 |
SRR24302025 | DROSHA_KO | full-length DROSHA | HCT116 | rep3 |
SRR24302026 | DROSHA_KO | full-length DROSHA | HCT116 | rep2 |
SRR24302027 | DROSHA_KO | full-length DROSHA | HCT116 | rep1 |
Download data
Prepare the following PBS Pro script to submit the request to download of files to the HPC cluster.
| Code Block |
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#!/bin/bash -l #PBS -N download_ENA_data #PBS -l select=1:ncpus=2:mem=8gb #PBS -l walltime=24:00:00 #work on current directory (folder) cd $PBS_O_WORKDIR wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/023/SRR24302023/SRR24302023.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/017/SRR24302017/SRR24302017.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/019/SRR24302019/SRR24302019.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/012/SRR24302012/SRR24302012.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/026/SRR24302026/SRR24302026.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/027/SRR24302027/SRR24302027.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/013/SRR24302013/SRR24302013.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/015/SRR24302015/SRR24302015.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/021/SRR24302021/SRR24302021.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/024/SRR24302024/SRR24302024.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/010/SRR24302010/SRR24302010.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/018/SRR24302018/SRR24302018.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/022/SRR24302022/SRR24302022.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/011/SRR24302011/SRR24302011.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/009/SRR24302009/SRR24302009.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/016/SRR24302016/SRR24302016.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/020/SRR24302020/SRR24302020.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/008/SRR24302008/SRR24302008.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/025/SRR24302025/SRR24302025.fastq.gz wget -nc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR243/014/SRR24302014/SRR24302014.fastq.gz |
Submit the download script to the cluster:
| Code Block |
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qsub launch_ENA_download.pbs |
Monitor progress of job:
| Code Block |
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qjobs |
Preparing “sample sheet” index file:
| Code Block |
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#fetch samples names
ls --color=never *.gz -1 | awk -F "." '{print $1}' > sample.names
#fetch the path to each FASTQ file
for i in `ls --color=never *.gz`; do echo $i; readlink -f $i >> sample.path ; done
#merge the sample names and FASTQ path
echo "sample,fastq_1" > samplesheet.csv
paste sample.names sample.path | awk '{print $1 "," $2}' >> samplesheet.csv
#remove intermediate files
rm sample.names sample.path |