Download public small RNA-see data

Owned by Roberto Barrero Gumiel
Last updated: Aug 09, 2023
2 min read

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.

#!/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:

qsub launch_ENA_download.pbs

Monitor progress of job:

qjobs

 

Preparing “sample sheet” index file: