GSE179634
GSE GEOSplicing Factor SRSF1 Deficiency in the Liver Triggers NASH-like Pathology via R-Loop Induced DNA Damage and Cell Death
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Summary
Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA-DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Accumulation of lipids in SRSF1-deficient hepatocytes is quickly followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. This pathogenesis is recapitulated in SRSF1-depleted human liver cancer cells illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver failure.
Overall Design
Hepatocytes were isolated from 1-month-old C57BL/6j (2 replicates) using the two-step collagenase perfusion method. Isolated hepatocytes were crosslinked with 400 mJ/cm2 of 254 nm UV radiation to stabilize RNA binding protein (RBP)-RNA interactions. Subsequent immunoprecipitation of SRSF1-RNA complexes, RNA isolation, library preparation and sequencing were performed using the eCLIP protocol described in the ENCODE project.
Analysis (32 steps)
View Data Processing- Takes output from raw files.
- Run to trim off both 5â and 3â adapters on both reads.
- Command: quality-cutoff 6 -m 18 -a NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -g CTTCCGATCTACAAGTT -g CTTCCGATCTTGGTCCT -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGT AGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz /full/path/to/files/file_R1.C01.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.metrics
- Takes output from cutadapt round 1.
- Run to trim off the 3â adapters on read 2, to control for double ligation events.
- Command: cutadapt -f fastq --match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGTAGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.metrics
- Takes output from cutadapt round 2.
- Maps to human specific version of RepBase used to remove repetitive elements, helps control for spurious artifacts from rRNA (& other) repetitive reads.
Supplementary Files (1)
GEO Samples (4)
SRA Experiments (4) and Runs (4)
Total: 1932 MBSample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR15058673 | 12846688 | 1413135680 | 530.58 | SRSF1.SRSF1_WT_CLIP_A.r1.reformatted.fq.gz, SRSF1.SRSF1_WT_CLIP_A.r2.… | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR15058674 | 10255093 | 1128060230 | 421.82 | SRR15058674.lite, SRR15058674, SRSF1.SRSF1_WT_CLIP_B.r2.reformatted.f… | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR15058675 | 11050104 | 1211960695 | 452.15 | SRSF1.SRSF1_WTA_INPUT.NIL.r2.fq.gz.addrandomer.fq.gz, SRSF1_WTA_INPUT… | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR15058676 | 12808221 | 1404476810 | 527.37 | SRSF1.SRSF1_WTB_INPUT.NIL.r2.fq.gz.addrandomer.fq.gz, SRSF1_WTB_INPUT… | SRA |
Linked Publications (1)
Data Files (4)
| Accession | File Name | Stored Type | Output Type | Mapping Assembly | Size | Download | |
|---|---|---|---|---|---|---|---|
| — | SRR15058674.lite | RIP-Seq | 421.8 MB | link | |||
| — | SRSF1.SRSF1_WTA_INPUT.NIL.r2.fq.gz.addrandomer.fq.gz | RIP-Seq | 452.1 MB | link | |||
| — | SRSF1.SRSF1_WTB_INPUT.NIL.r2.fq.gz.addrandomer.fq.gz | RIP-Seq | 527.4 MB | link | |||
| — | SRSF1.SRSF1_WT_CLIP_A.r1.reformatted.fq.gz | RIP-Seq | 530.6 MB | link |