A CRISPR RNA-binding protein screen reveals regulators of RUNX1 isoform generation.
Amanda G Davis, Jaclyn M Einstein, Dinghai Zheng, Nathan D Jayne, Xiang-Dong Fu, Bin Tian, Gene W Yeo, Dong-Er Zhang
Blood advances · 2021 · Vol. 5 (5) · pp. 1310-1323
Abstract
The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and is disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined central role, and mutations promote malignant transformation. More recently, studies have illuminated the importance of posttranscriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and the breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between posttranscriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the posttranscriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a vs RUNX1b/c) that mediate HSC self-renewal vs differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detect changes in RNA processing. We used this reporter in a clustered regularly interspaced short palindromic repeats (CRISPR) screen consisting of single guide RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the posttranscriptional regulation of a key hematopoietic transcription factor and identifies RBPs that may have widespread and important functions in hematopoiesis.
Linked datasets 1
CRISPR/Cas9 screening of RNA binding proteins (RBPs) that regulate RUNX1 isoform production
Homo sapiens
22 data files
| File | Type | Size |
|---|---|---|
| CMP_Batch_2_S3_L008_R1_001.fastq.gz | RNA-Seq | 1001.8 MB |
| CMP_Batch_4_S4_L008_R1_001.fastq.gz | RNA-Seq | 825.6 MB |
| CMP_Batch_5_S5_L008_R1_001.fastq.gz | RNA-Seq | 715.4 MB |
| MD7a__1_GFP_High_S3_L007_R1_001.fastq.gz | OTHER | 158.4 MB |
| MD7a__1_GFP_Low_S4_L007_R1_001.fastq.gz | OTHER | 134.5 MB |
| MD7a__1_t_0_S1_L007_R1_001.fastq.gz | OTHER | 312.0 MB |
| MD7a__1_t_end_S2_L007_R1_001.fastq.gz | OTHER | 329.6 MB |
| MD7a__2_GFP_High_S7_L007_R1_001.fastq.gz | OTHER | 141.5 MB |
| MD7a__2_GFP_Low_S8_L007_R1_001.fastq.gz | OTHER | 142.0 MB |
| MD7a__2_t_0_S5_L007_R1_001.fastq.gz | OTHER | 280.3 MB |
| MD7a__2_t_end_S6_L007_R1_001.fastq.gz | OTHER | 297.1 MB |
| SRR11185549_GSM4340380 | OTHER | 158.0 MB |
| SRR11185550_GSM4340381 | OTHER | 141.0 MB |
| SRR11185551_GSM4340382 | OTHER | 134.0 MB |
| SRR11185552_GSM4340383 | OTHER | 142.0 MB |
| SRR11185553_GSM4340384 | OTHER | 329.0 MB |
| SRR11185554_GSM4340385 | OTHER | 297.0 MB |
| SRR11185555_GSM4340386 | OTHER | 312.0 MB |
| SRR11185556_GSM4340387 | OTHER | 280.0 MB |
| SRR11185557_GSM4340388 | RNA-Seq | 1001.0 MB |
| SRR11185558_GSM4340389 | RNA-Seq | 825.0 MB |
| SRR11185559_GSM4340390 | RNA-Seq | 715.0 MB |
Analysis pipelines 1
| Pipeline | Assay | Steps | Dataset |
|---|---|---|---|
| GSE145968 RNA-seq Data Processing geo_data_processing | RNA-seq | 6 | GSE145968 |
Funding 10 grants
| Grant | Funder |
|---|---|
| R01 HG004659 | NHGRI NIH HHS (United States) |
| U54 CA209891 | NCI NIH HHS (United States) |
| T32 GM007240 | NIGMS NIH HHS (United States) |
| P50 GM085764 | NIGMS NIH HHS (United States) |
| R01 DK098808 | NIDDK NIH HHS (United States) |
| F31 CA217173 | NCI NIH HHS (United States) |
| F31 HL131155 | NHLBI NIH HHS (United States) |
| P30 CA010815 | NCI NIH HHS (United States) |
| R01 GM129069 | NIGMS NIH HHS (United States) |
| R01 GM084089 | NIGMS NIH HHS (United States) |