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CRISPR-assisted RNA-protein interaction detection (CARPID) combined with quantitative mass spectrometry was used to identify proteins interacting with the long non-coding RNA (lncRNA) Taurine Up-regulated 1 (TUG1) in HEK293T cells under physiological conditions and after Camptothecin (CPT) treatment.

ABSTRACT: This project utilized CRISPR-assisted RNA-protein interaction detection (CARPID) combined with mass spectrometry to identify proteins interacting with the long non-coding RNA (lncRNA) TUG1 in HEK293T cells. TUG1 preferentially interacts with known R-loop-associated proteins, particularly under Camptothecin (CPT) treatment. Additionally, we identified interacting proteins for the lncRNAs MALAT1 to validate the binding specificity of TUG1.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yutaka Kondo

PROVIDER: PXD049137 | JPOST Repository | Thu Jul 17 00:00:00 GMT+01:00 2025

REPOSITORIES: jPOST

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Comprehensive identification of proteins interacting with long non-coding RNA TUG1 in R-loop regulation.

Xie Jingqi J Suzuki Miho M MM Iijima Kenta K Shinjo Keiko K Nishimura Tatsunori T Watanabe Shinya S Nakagawa Reiko R Ito Tatsuo T Kondo Yutaka Y

Journal of biochemistry 20250901 4

Long non-coding RNAs (lncRNAs) regulate a wide array of cellular processes through interactions with RNA-binding proteins (RBPs). Taurine Upregulated Gene 1 (TUG1) is an lncRNA that is overexpressed in many types of cancer and has been implicated in resolving R-loops, thereby maintaining genomic integrity. However, the full spectrum of its protein interactions and stress-responsive dynamics remains unclear. Here, we employed CRISPR-assisted RNA-protein interaction detection (CARPID) combined wit ...[more]

PMID: 40654306

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Project description:Neuroblastoma (NB) is an embryonal tumor with various clinical presentations and behaviors. Several genomic alterations has been well-studied in NB, among which genomic amplification of MYCN oncogene, is a strong prognostic biomarker with worsens outcome. Long noncoding RNAs (lncRNAs), constitute major proportion of the cellular transcripts with no coding capacity. One of their function is to guide transcription factors to the target genes and facilitate gene expression. However, relative contribution of lncRNA and MYCN to the advanced NB has remained unclear. Herein, by applying a network-based integrative analysis on MYCN amplified and MYCN nonamplified lncRNA expression profile from both RNA-seq and microarray platform, we identified lncRNA, SNHG1 to be differentially expressed and strongly correlated with MYCN in MYCN-amplified NB. The expression of SNHG1 was validated by RT-qPCR in NB cell lines. Survival analysis revealed that higher expression of SNHG1 significantly associates with poor patient survival. Moreover, knockdown of MYCN in MYCN-amplified NB cell lines inhibited SNHG1 expression. Furthermore, to unravel the role of SNHG1 in NB, we extracted SNHG1-interacting proteins by RNA-protein pull down assay coupled with doi:10.6342/NTU201701980 ! ! VI liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 27 SNHG1-interacting proteins in common from three NB cell lines. However, only three SNHG1-interacting proteins, MATR3, YBX1 and HHRNPL have binding site detected by DeepBind motif analysis. Western blot confirms interaction of MATR3 with SNHG1. Additionally, we further validated the direct interaction between MATR3 and SNHG1 by RNA-immunoprecipation (IP). MATR3 is known to be involved in RNA transport and stabilization. Therefore, we proposed that MATR3 after interacting with SNHG1 might help in SNHG1 transcription and stabilization. In conclusion, our study unveils that SNHG1 could be a prognostic marker for high-risk NB and possibly stabilized by MATR3. Our results might provide future directions for the development of therapeutic strategies against high-risk NB.

Project description:BackgroundTuberculosis (TB) remains a major public health problem. Long non-coding RNAs (lncRNAs) are important regulators of gene expression. In this study, we explored the association between the expression of lncRNA AC007128.1 and TB susceptibility.MethodsThree single-nucleotide polymorphisms (SNPs) (rs12333784, rs6463794, and rs720964) of lncRNA AC007128.1 were selected using the 1000 Genomes Project database and offline software Haploview V4.2, and were genotyped by a customized 2×48-Plex SNPscan™ Kit.ResultsWe identified two differentially expressed lncRNA including AC007128.1 and AP001065.3 in comparisons of expression profiles between ATB vs. LTBI, LTBI vs. HCs, and AC700128.1 expression was specifically and significantly up-regulated in TB patients by verification of external data. Gene Ontology functional enrichment analysis and co-expression network showed up-regulated mRNA was mainly involved in negative regulation of the G protein-coupled receptor (GPCR) signaling pathway, and FPR1 and CYP27B1 were involved in the co-expression of AC007128.1. Using the 1000 Genomes Project, software Haploview V4.2, and SNP genotype, we screened out SNP rs12333784 which locus at 7p21.3 in AC007128.1 associated with TB susceptibility. The G carrier of rs12333784 was then finally verified to be significantly associated with pulmonary TB (PTB) and extrapulmonary tuberculosis (EPTB) susceptibility (pBonferroni =0.03878), and a similar but more significant effect was observed under the dominant model analysis (pBonferroni =0.013, OR =1.349, 95% CI, 1.065-1.709). In addition, the GG + GA genotype of SNP rs12333784 was significantly correlated with higher glucose (GLU) (P=0.03), higher gamma-glutamyl transferase (GGT) (P=0.05), and higher erythrocyte sedimentation rate (ESR) (P=0.05).ConclusionsOur findings show lncRNA AC007128.1 can be regarded as biomarkers discriminating between ATB and LTBI and may also be a diagnostic biomarker for LBTI. These findings may aid clinical decision making in the management of TB.

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Publications

Comprehensive identification of proteins interacting with long non-coding RNA TUG1 in R-loop regulation.

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