CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer
Caishang Zheng, Yanjun Wei, Peng Zhang, Longyong Xu, Zhenzhen Zhang, Kangyu Lin, Jiakai Hou, Xiangdong Lv, Yao Ding, Yulun Chiu, Antrix Jain, Nelufa Islam, Anna Malovannaya, Yun Wu, Feng Ding, Han Xu, Ming Sun, Xi Chen, Yiwen Chen
Caishang Zheng, Yanjun Wei, Peng Zhang, Longyong Xu, Zhenzhen Zhang, Kangyu Lin, Jiakai Hou, Xiangdong Lv, Yao Ding, Yulun Chiu, Antrix Jain, Nelufa Islam, Anna Malovannaya, Yun Wu, Feng Ding, Han Xu, Ming Sun, Xi Chen, Yiwen Chen
View: Text | PDF
Research Article Genetics Oncology

CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer

Abstract

Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α–positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the “hidden” lncRNA-encoded proteome might be a new space for therapeutic target discovery.

Authors

Caishang Zheng, Yanjun Wei, Peng Zhang, Longyong Xu, Zhenzhen Zhang, Kangyu Lin, Jiakai Hou, Xiangdong Lv, Yao Ding, Yulun Chiu, Antrix Jain, Nelufa Islam, Anna Malovannaya, Yun Wu, Feng Ding, Han Xu, Ming Sun, Xi Chen, Yiwen Chen

×

Figure 9

GT3-INCP is upregulated by estrogen/ER and is important for estrogen-dependent cell growth/gene expression.

Options: View larger image (or click on image) Download as PowerPoint
GT3-INCP is upregulated by estrogen/ER and is important for estrogen-dep...
(A) qRT-PCR analysis of LINC00992 RNA expression and (B) Western blot analysis of GT3-INCP expression in MCF7 and T47D cells upon β-estradiol (E2) treatment (30 nM) for the indicated time intervals. Western blot analysis of GT3-INCP expression (C) in MCF7/T47D cells transfected with ESR1-targeting endoribonuclease-prepared siRNA (esiRNA) or GFP-targeting esiRNA EGFP (esiEGFP), or (D) in the cells treated with 15 μM 4-hydroxytamoxifen (4-OHT; Sigma-Aldrich, SML1666) or vehicle (ethanol, ETOH) control. (E) qRT-PCR analysis of LINC00992 RNA expression in MCF7 cells that were transfected with the negative control siRNA (siNC) or LINC00992-targeting siRNAs (siLINC00992), after E2 (30 nM) or ETOH vehicle treatment. (F) After E2/ETOH treatment, the numbers of MCF7 cells treated with transfection reagent (control) or transfected with siNC or GATA3-targeting siRNAs (siGATA3) or siLINC00992 were counted every 24 hours for 72 hours. (G) After E2/ETOH treatment, the number of MCF7 cells that were treated with transfection reagent (control) or the MCF7 cells that were transduced with the empty vector (EV) or the indicated ORFs and transfected with siNC/siLINC00992 (siL) was monitored for 72 hours. (H) qRT-PCR analysis of MYB and PDZK1 RNA expression in MCF7 cells that were transfected with siNC, siGATA3, or siLINC00992, after E2/ETOH treatment. qRT-PCR analysis of (I) MYB and (J) PDZK1 RNA expression in the MCF7 cells that were treated with transfection reagent (control) or the MCF7 cells that were transduced with EV or the indicated ORFs and transfected with siNC/LINC00992-targeting siRNA (siL), after E2/ETOH treatment. Data in A and E–J are shown as mean ± SD (n = 3). **P < 0.01 by 1-way ANOVA with Dunnett’s multiple-comparison test. NS, not significant (P > 0.05). Data in B–D are representative of 3 independent experiments.