Abstract
Long noncoding RNAs (lncRNAs) have recently been widely recognized as critical epigenetic regulators, with their function and stability dictated by the interactions with RNA-binding proteins (RBPs). However, current methodologies for studying lncRNA-protein interactions face significant technical limitations, such as reliance on chemical crosslinking and the availability of commercial antibodies. To overcome these challenges, we developed CRISPR-assisted RNA-protein interaction detection (CARPID) method, which leverages CRISPR/dCasRx-based RNA targeting and proximity labeling to identify binding proteins of specific endogenous lncRNAs in native cellular context. Applying CARPID to the nuclear lncRNA XIST successfully captured both known and previously uncharacterized binding proteins. We further generalized CARPID to explore binders of the lncRNAs DANCR and MALAT1, revealing the method’s wide applicability in identifying RBPs.To continue to enhance the capability of CARPID in challenging-to-transfect cells, we developed CARPID 2.0 by integrating the ribonucleoprotein (RNP)-based delivery system. Additionally, we replaced the CRISPR/dCasRx and biotin ligase BASU with the compact RNA-targeting miniCas13X.1 and engineered peroxidase APEX2, respectively. This upgrade enabled ultra-rapid labelling of adjacent proteins and RNAs within one minute, remarkably reducing non-specific background noise caused by prolonged reaction time. CARPID 2.0 has proven effective across various cell types, including neuronal and cancer cells, facilitating comprehensive identification of RBPs of endogenous lncRNAs.
We applied CARPID 2.0 to investigate the functional role of human endogenous retrovirus subfamily H (HERV-H), a group of transcriptionally active primate-specific retrotransposon-derived lncRNA associated with the pluripotency of stem cells. We identified 252 proteins significantly associated with HERV-H RNA, with over 70% involved in the process of ribosome biogenesis. Concurrently, ChIRP-seq analysis revealed that HERV-H RNA extensively binds to repeated intergenic space region (IGS) of ribosomal DNA (rDNA) clusters, which are responsible for the expression of pre-rRNA. Using our newly developed lncR-SELEX, and a series of electrophoretic mobility shift assay (EMSA), circular dichroism (CD), thermal UV melting, and nuclear magnetic resonance (NMR) assays, which enable in vitro profiling of RNA-DNA interactions, we discovered that HERV-H RNA be able to form trans-acting anti-parallel RNA:dsDNA triplex structure in IGS region via non-canonical base pairing. Consistently, we found that depletion of HERV-H RNA led to downregulation of pre-rRNA transcription, and perturbation of HERV-H RNA associated proteins identified 47 proteins as positive regulators of pre-rRNA expression.
To clarify this regulatory network, we employed a CRISPR/dCas9-assisted BioID strategy (dCas9-BASU) targeting IGS regions, identifying 173 significantly enriched proximal proteins. Notably, five of them, HMGA1, TCOF1, TOP1, SUB1, and PCBP2, were non-ribosomal proteins also found among HERV-H RNA-dependent positive regulators of pre-rRNA expression. Next, proteomic analysis of HERV-H RNA-depleted hESCs revealed significant downregulation of 103 proteins involved in RNA metabolism, ribosome biogenesis, and stem cell population maintenance, which was highly correlated with the role of HERV-H RNA in pre-rRNA transcription and stemness maintenance. Among them, PRDM14 protein was the most markedly affected, despite unchanged mRNA levels, underscoring its dependence on robust translational capacity for stemness maintenance. Collectively, our findings uncovered a novel mechanism of HERV-H RNA-mediated stemness maintenance through the regulation of pre-rRNA expression and ribosome biogenesis, a process crucial for the high demand of pluripotency-relevant factors in rapidly proliferating stem cell.
| Date of Award | 12 Aug 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Jian YAN (Supervisor) |
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