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

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

doi:10.1172/jci159940

Cited by 14 publications

(26 citation statements)

References 68 publications

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“…First, ribo-seq was performed to map the translatome in the CRC cell line HCT-116 as described previously 7 , 15 (Fig. 1a and the Methods ).…”

Section: Resultsmentioning

confidence: 99%

“…1c,d and Supplementary Table 1 ). The screen was then performed in HCT-116 cells stably expressing wild-type Streptococcus pyogenes Cas9 (SpCas9), following a protocol akin to those in our previous studies 15 , 20 – 22 . In brief, cells were transduced with lentiviral vectors containing the sgRNA library and subsequently subjected to puromycin selection.…”

Section: Resultsmentioning

confidence: 99%

“…Despite increasing appreciation of the functional importance of cryptic non-canonical ORFs in development, physiology and disease, the role and functional mechanism of human cryptic non-canonical ORFs in complex diseases such as cancer remain largely unknown. To fill this gap, we applied an integrative multiomic strategy 15 combining ribo-seq, a CRISPR–Cas9 pooled screen 16 with computational analysis of the Cancer Genome Atlas (TCGA) 17 and/or Genotype–Tissue Expression (GTEx) 18 data to identify cryptic ORFs that may be functionally important and potentially clinically relevant in CRC, the third most commonly diagnosed cancer in men, the second most common cancer in women and the second mostly deadly cancer worldwide. We further characterized the function and mechanism of a cryptic ELFN1 - AS1 -encoded microprotein that was identified from our screen.…”

Section: Mainmentioning

confidence: 99%

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CRISPR–Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames

Zheng,

Wei,

Zhang

et al. 2023

Nat Struct Mol Biol

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Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR–Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the ‘dark’ proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

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“…First, ribo-seq was performed to map the translatome in the CRC cell line HCT-116 as described previously 7 , 15 (Fig. 1a and the Methods ).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Mainmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR–Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames

Zheng,

Wei,

Zhang

et al. 2023

Nat Struct Mol Biol

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“…Although more research is needed in this area, dORF translation may enhance main CDS translation ( 187 ), although dORF translation is, in general, very rare, likely occurring on <5% of mRNA transcripts ( 8 , 160 ). ORFs found in long non-coding RNAs (lncRNAs) are also likely to produce targetable cancer antigens ( 156 , 158 ), but these ORFs also are better described as a source for novel cancer microproteins with biological roles in disease pathophysiology ( 140 , 188 ).…”

Section: Beyond Uorfs: Other Non-canonical Orfs In Cancermentioning

confidence: 99%

Upstream open reading frames: new players in the landscape of cancer gene regulation

Dasgupta,

Prensner

2024

NAR Cancer

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The translation of RNA by ribosomes represents a central biological process and one of the most dysregulated processes in cancer. While translation is traditionally thought to occur exclusively in the protein-coding regions of messenger RNAs (mRNAs), recent transcriptome-wide approaches have shown abundant ribosome activity across diverse stretches of RNA transcripts. The most common type of this kind of ribosome activity occurs in gene leader sequences, also known as 5′ untranslated regions (UTRs) of the mRNA, that precede the main coding sequence. Translation of these upstream open reading frames (uORFs) is now known to occur in upwards of 25% of all protein-coding genes. With diverse functions from RNA regulation to microprotein generation, uORFs are rapidly igniting a new arena of cancer biology, where they are linked to cancer genetics, cancer signaling, and tumor-immune interactions. This review focuses on the contributions of uORFs and their associated 5′UTR sequences to cancer biology.

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“…A few microproteins, such as phospholamban (PLN, 55 aa), sarcolipin (SLN, 31 aa), and myoregulin (MRLN, 46 aa), regulate sarcoplasmic reticulum Ca 2+ ATPase activity and muscle function 26 – 28 . A putative human lncRNA LINC00992 encodes a GATA3-interacting cryptic protein (GT3-INCP, 120 aa) that promotes estrogen receptor-positive breast cancer progression 29 .…”

Section: Introductionmentioning

confidence: 99%

Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features

Yang,

Li,

Stroup

et al. 2024

Nat Commun

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Studies have revealed dozens of functional peptides in putative ‘noncoding’ regions and raised the question of how many proteins are encoded by noncanonical open reading frames (ORFs). Here, we comprehensively annotate genome-wide translated ORFs across five eukaryotes (human, mouse, zebrafish, worm, and yeast) by analyzing ribosome profiling data. We develop a logistic regression model named PepScore based on ORF features (expected length, encoded domain, and conservation) to calculate the probability that the encoded peptide is stable in humans. Systematic ectopic expression validates PepScore and shows that stable complex-associating microproteins can be encoded in 5’/3’ untranslated regions and overlapping coding regions of mRNAs besides annotated noncoding RNAs. Stable noncanonical proteins follow conventional rules and localize to different subcellular compartments. Inhibition of proteasomal/lysosomal degradation pathways can stabilize some peptides especially those with moderate PepScores, but cannot rescue the expression of short ones with low PepScores suggesting they are directly degraded by cellular proteases. The majority of human noncanonical peptides with high PepScores show longer lengths but low conservation across species/mammals, and hundreds contain trait-associated genetic variants. Our study presents a statistical framework to identify stable noncanonical peptides in the genome and provides a valuable resource for functional characterization of noncanonical translation during development and disease.

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

Cited by 14 publications

References 68 publications

CRISPR–Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames

CRISPR–Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames

Upstream open reading frames: new players in the landscape of cancer gene regulation

Widespread stable noncanonical peptides identified by integrated analyses of ribosome profiling and ORF features

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