Integrative CRISPR Screening and RNA Analyses Discover an Essential Role for PUF60 Interactions with 3' Splice Sites in Cancer Progression.
Alexandra T Tankka, Yuhan Zhang, Jaclyn M Einstein, Catherine J Zhou, Vivian N Pham, Jack T Naritomi, Grady G Nguyen, Amaya N Mendez-Molina, Zhimin Hu, Orel Mizrahi, Mark Perelis, Joseph Sarsam, Frederick E Tan, Dan S Kaufman, Jing Yang, Corina E Antal, Gene W Yeo
Cancer research · 2025
Abstract
RNA-binding proteins (RBPs) are important regulators of post-transcriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies, such as triple negative breast cancer (TNBC). Here, we employed both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates essential for TNBC cell survival. Integrated eCLIP and RNA-sequencing analysis identified that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when mis-spliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3' splice sites via a substitution in its RNA-binding domain caused widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. Knockdown of PUF60 or disruption of PUF60-RNA interactions inhibited TNBC cell proliferation and shrunk tumor xenografts in multiple models. Together, these findings reveal the molecular mechanism by which PUF60 supports cancer progression.
Linked datasets 4
PUF60-Mediated Splicing Is a Key Driver of Triple Negative Breast Cancer [RNA-seq]
40 data files
| File | Type | Size |
|---|---|---|
| SRR31256676_GSM8607263 | RNA-Seq | 4.7 GB |
| SRR31256677_GSM8607262 | RNA-Seq | 4.7 GB |
| SRR31256678_GSM8607261 | RNA-Seq | 4.4 GB |
| SRR31256679_GSM8607260 | RNA-Seq | 8.4 GB |
| SRR31256680_GSM8607259 | RNA-Seq | 5.0 GB |
| SRR31256681_GSM8607258 | RNA-Seq | 5.7 GB |
| SRR31256682_GSM8607257 | RNA-Seq | 5.9 GB |
| SRR31256683_GSM8607256 | RNA-Seq | 4.4 GB |
| SRR31256684_GSM8607255 | RNA-Seq | 6.1 GB |
| SRR31256685_GSM8607254 | RNA-Seq | 4.0 GB |
| SRR31256686_GSM8607253 | RNA-Seq | 4.7 GB |
| SRR31256687_GSM8607252 | RNA-Seq | 5.2 GB |
| SRR31256688_GSM8607251 | RNA-Seq | 4.6 GB |
| SRR31256689_GSM8607250 | RNA-Seq | 6.5 GB |
| SRR31256690_GSM8607249 | RNA-Seq | 5.9 GB |
| SRR31256691_GSM8607248 | RNA-Seq | 5.1 GB |
| SRR31256692_GSM8607247 | RNA-Seq | 6.3 GB |
| SRR31256693_GSM8607246 | RNA-Seq | 4.4 GB |
| SRR31256694_GSM8607245 | RNA-Seq | 5.1 GB |
| SRR31256695_GSM8607244 | RNA-Seq | 5.6 GB |
| SRR31256696_GSM8607243 | RNA-Seq | 5.1 GB |
| SRR31256697_GSM8607242 | RNA-Seq | 6.3 GB |
| SRR31256698_GSM8607241 | RNA-Seq | 7.8 GB |
| SRR31256699_GSM8607240 | RNA-Seq | 7.0 GB |
| SRR31256700_GSM8607239 | RNA-Seq | 9.0 GB |
| SRR31256701_GSM8607238 | RNA-Seq | 8.9 GB |
| SRR31256702_GSM8607237 | RNA-Seq | 4.3 GB |
| SRR31256703_GSM8607236 | RNA-Seq | 6.1 GB |
| SRR31256704_GSM8607235 | RNA-Seq | 4.2 GB |
| SRR31256705_GSM8607234 | RNA-Seq | 6.0 GB |
| SRR31256706_GSM8607233 | RNA-Seq | 6.3 GB |
| SRR31256709_GSM8607230 | RNA-Seq | 5.5 GB |
| SRR31256710_GSM8607229 | RNA-Seq | 5.2 GB |
| SRR31268945_GSM8617980 | RNA-Seq | 4.6 GB |
| SRR31268946_GSM8617979 | RNA-Seq | 5.2 GB |
| SRR31268947_GSM8617978 | RNA-Seq | 4.8 GB |
| SRR31268948_GSM8617977 | RNA-Seq | 6.6 GB |
| SRR31268949_GSM8617976 | RNA-Seq | 3.2 GB |
| SRR36433264_GSM8607231 | RNA-Seq | 7.2 GB |
| SRR36433265_GSM8607232 | RNA-Seq | 6.8 GB |
PUF60-Mediated Splicing Is a Key Driver of Triple Negative Breast Cancer [eCLIP]
26 data files
| File | Type | Size |
|---|---|---|
| SRR31195360_GSM8607292 | RIP-Seq | 220.0 MB |
| SRR31195362_GSM8607291 | RIP-Seq | 350.0 MB |
| SRR31195364_GSM8607290 | RIP-Seq | 683.0 MB |
| SRR31195366_GSM8607289 | RIP-Seq | 658.0 MB |
| SRR31195368_GSM8607288 | RIP-Seq | 580.0 MB |
| SRR31195370_GSM8607287 | RIP-Seq | 610.0 MB |
| SRR31195372_GSM8607286 | RIP-Seq | 709.0 MB |
| SRR31195374_GSM8607285 | RIP-Seq | 764.0 MB |
| SRR31195376_GSM8607284 | RIP-Seq | 1.1 GB |
| SRR31195378_GSM8607283 | RIP-Seq | 1.3 GB |
| SRR31195380_GSM8607282 | RIP-Seq | 669.0 MB |
| SRR31195382_GSM8607281 | RIP-Seq | 529.0 MB |
| SRR31195384_GSM8607280 | RIP-Seq | 280.0 MB |
| SRR31195386_GSM8607279 | RIP-Seq | 347.0 MB |
| SRR31195388_GSM8607278 | RIP-Seq | 744.0 MB |
| SRR31195390_GSM8607277 | RIP-Seq | 596.0 MB |
| SRR31195392_GSM8607276 | RIP-Seq | 726.0 MB |
| SRR31195394_GSM8607275 | RIP-Seq | 582.0 MB |
| SRR31195396_GSM8607274 | RIP-Seq | 500.0 MB |
| SRR31195398_GSM8607273 | RIP-Seq | 558.0 MB |
| SRR31195400_GSM8607272 | RIP-Seq | 992.0 MB |
| SRR31195402_GSM8607271 | RIP-Seq | 900.0 MB |
| SRR31195404_GSM8607270 | RIP-Seq | 2.4 GB |
| SRR31195406_GSM8607269 | RIP-Seq | 2.5 GB |
| SRR31195408_GSM8607268 | RIP-Seq | 763.0 MB |
| SRR31195410_GSM8607267 | RIP-Seq | 515.0 MB |
PUF60-Mediated Splicing Is a Key Driver of Triple Negative Breast Cancer
Homo sapiens
PUF60-Mediated Splicing Is a Key Driver of Triple Negative Breast Cancer [CRISPR]
46 data files
| File | Type | Size |
|---|---|---|
| SRR36398575_GSM9366239 | OTHER | 119.0 MB |
| SRR36398576_GSM9366238 | OTHER | 124.0 MB |
| SRR36398577_GSM9366237 | OTHER | 125.0 MB |
| SRR36398578_GSM9366236 | OTHER | 130.0 MB |
| SRR36398579_GSM9366235 | OTHER | 96.0 MB |
| SRR36398580_GSM9366234 | OTHER | 113.0 MB |
| SRR36398581_GSM9366233 | OTHER | 105.0 MB |
| SRR36398582_GSM9366232 | OTHER | 123.0 MB |
| SRR36398583_GSM9366231 | OTHER | 126.0 MB |
| SRR36398584_GSM9366230 | OTHER | 107.0 MB |
| SRR36398585_GSM9366229 | OTHER | 119.0 MB |
| SRR36398586_GSM9366228 | OTHER | 108.0 MB |
| SRR36398587_GSM9366227 | OTHER | 119.0 MB |
| SRR36398588_GSM9366226 | OTHER | 129.0 MB |
| SRR36398589_GSM9366225 | OTHER | 109.0 MB |
| SRR36398590_GSM9366224 | OTHER | 125.0 MB |
| SRR36398591_GSM9366223 | OTHER | 105.0 MB |
| SRR36398592_GSM9366222 | OTHER | 123.0 MB |
| SRR36398593_GSM9366221 | OTHER | 120.0 MB |
| SRR36398594_GSM9366220 | OTHER | 97.0 MB |
| SRR36398595_GSM9366219 | OTHER | 166.0 MB |
| SRR36398596_GSM9366218 | OTHER | 119.0 MB |
| SRR36398597_GSM9366217 | OTHER | 164.0 MB |
| SRR36398598_GSM9366216 | OTHER | 168.0 MB |
| SRR36398599_GSM9366215 | OTHER | 85.0 MB |
| SRR36398600_GSM9366214 | OTHER | 183.0 MB |
| SRR36398601_GSM9366213 | OTHER | 147.0 MB |
| SRR36398602_GSM9366212 | OTHER | 179.0 MB |
| SRR36398603_GSM9366211 | OTHER | 167.0 MB |
| SRR36398604_GSM9366210 | OTHER | 136.0 MB |
| SRR36398605_GSM9366209 | OTHER | 46.0 MB |
| SRR36398606_GSM9366208 | OTHER | 49.0 MB |
| SRR36398607_GSM9366207 | OTHER | 49.0 MB |
| SRR36398608_GSM9366206 | OTHER | 50.0 MB |
| SRR36398609_GSM9366205 | OTHER | 61.0 MB |
| SRR36398610_GSM9366204 | OTHER | 52.0 MB |
| SRR36398611_GSM9366203 | OTHER | 48.0 MB |
| SRR36398612_GSM9366202 | OTHER | 62.0 MB |
| SRR36398613_GSM9366201 | OTHER | 44.0 MB |
| SRR36398614_GSM9366200 | OTHER | 39.0 MB |
| SRR36398615_GSM9366199 | OTHER | 34.0 MB |
| SRR36398616_GSM9366198 | OTHER | 49.0 MB |
| SRR36398617_GSM9366197 | OTHER | 820.0 MB |
| SRR36398618_GSM9366196 | OTHER | 871.0 MB |
| SRR36398619_GSM9366195 | OTHER | 814.0 MB |
| SRR36398620_GSM9366194 | OTHER | 833.0 MB |
Potentially related datasets 1
These accessions were text-mined from the PMC full text. They may be referenced for comparison, cited from other studies, or otherwise mentioned without being primary data for this paper.
Analysis pipelines 4
| Pipeline | Assay | Steps | Dataset |
|---|---|---|---|
| GSE280897 RNA-seq Data Processing geo_data_processing | RNA-seq | 6 | GSE280897 |
| GSE280898 eCLIP Data Processing geo_data_processing | eCLIP | 5 | GSE280898 |
| GSE280899 Data Processing geo_data_processing | — | 6 | GSE280899 |
| GSE313324 Data Processing geo_data_processing | — | 2 | GSE313324 |
Funding 14 grants
| Grant | Funder |
|---|---|
| R01 CA268179 | NCI NIH HHS (United States) |
| S10 OD025060 | NIH HHS (United States) |
| U24 HG009889 | NHGRI NIH HHS (United States) |
| S10 OD026929 | NIH HHS (United States) |
| R01 HG004659 | NHGRI NIH HHS (United States) |
| F32 HL143978 | NHLBI NIH HHS (United States) |
| P30 CA023100 | NCI NIH HHS (United States) |
| U54 CA209891 | NCI NIH HHS (United States) |
| R01 CA174869 | NCI NIH HHS (United States) |
| U24 HG011735 | NHGRI NIH HHS (United States) |
| RF1 MH126719 | NIMH NIH HHS (United States) |
| R01 CA262794 | NCI NIH HHS (United States) |
| R01 HG011864 | NHGRI NIH HHS (United States) |
| T32 CA067754 | NCI NIH HHS (United States) |