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
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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

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Figure 8

GT3-INCP facilitates the binding of GATA3 to the common cis regulatory elements of MYB and PDZK1.

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GT3-INCP facilitates the binding of GATA3 to the common cis regulatory e...
(A) The GT3-INCP and GATA3 ChIP-seq signal and peaks around MYB and PDZK1 in MCF7 and T47D cells. ChIP-qPCR validation of (B) GT3-INCP and (C) GATA3 binding to the ChIP-seq peaks around MYB and PDZK1 with the indicated antibodies in MCF7 cells stably expressing the FLAG-tagged GT3-INCP. (D) ChIP-qPCR analysis for assessing the effect of GT3-INCP knockout on GATA3 occupancy on its binding sites around MYB and PDZK1 in MCF7 cells. (E) Upon LINC00992 knockdown, ChIP-qPCR analysis was performed to assess the rescue effect of ectopic expression of wild-type GT3-INCP or mutant GT3-INCP (Del-M8 or AGG), with respect to the EV control, on the GATA3 occupancy on its binding sites in MCF7 cells. Data in B–E are shown as mean ± SD (n = 3). **P < 0.01 by 2-tailed, unpaired Student’s t test (B and C) or 1-way ANOVA with Dunnett’s multiple-comparison test (D and E). NS, not significant (P > 0.05).