CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer
Caishang Zheng, … , Xi Chen, Yiwen Chen
Caishang Zheng, … , Xi Chen, Yiwen Chen
Published March 1, 2023
Citation Information: J Clin Invest. 2023;133(5):e159940. https://doi.org/10.1172/JCI159940.
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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 3

LINC00992 encodes an unannotated protein.

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LINC00992 encodes an unannotated protein. (A) Ribo-seq count profile of...
(A) Ribo-seq count profile of 3 replicates across the LINC00992-encoded ORF. The predicted ORF based on GENCODE v22 annotation (ENST00000504107.1) is labeled “original predicted ORF” and the ORF with the extended region identified by 5′ RACE is labeled “extended new ORF.” (B) Schematic of LINC00992 gene and transcript (ENST00000504107.2, GENCODE v39) structure, and the information about its encoded protein GT3-INCP. (C) In the presence/absence of the native 5′-UTR, the wild-type FLAG-tagged GT3-INCP or the mutant one (AGG mutation in start codon) was stably expressed in MCF7, T47D, and ZR75-1 cells and protein expression was determined by Western blot with anti-FLAG and anti–GT3-INCP antibodies, where β-actin was used as a loading control. (D) Endogenous GT3-INCP protein expression was determined by Western blot in the indicated ER+ BC cell lines that were transduced with the negative control sgRNA (sgNC) or gene-specific sgRNAs, where β-actin served as a loading control. (E) The regions of GT3-INCP with the MS-identified peptides from IP of both ectopic FLAG-tagged and endogenous GT3-INCP in ER+ BC cells are shown in green and the corresponding sequences are shown in red. (F–I) The MS2 spectra of the GT3-INCP–derived tryptic peptides QERFPIILLSR and TDSFAGHLFSTAR detected by PRM-MS in the proteins coimmunoprecipitated with the anti-FLAG antibody from MCF7 (F and G) and T47D (H and I) cell lysates. Data in C and D are representative of 3 independent experiments.