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Identification of gene expression in cells that may express micropeptide from RN7SL1

ABSTRACT: Identification of gene expression in cells that may express micropeptide from RN7SL1

PROVIDER: PRJNA999352 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25442987.fastq.gz	Fastqsanger.gz
	SRR25442988.fastq.gz	Fastqsanger.gz
	SRR25442989.fastq.gz	Fastqsanger.gz
	SRR25442990.fastq.gz	Fastqsanger.gz

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Identification of gene expression in cells that may express micropeptide from RN7SL1

Project description:RN7SL1 is a 299-base single-stranded RNA that binds to five SRP proteins and contributes to the generation of transmembrane and secretory proteins as a signal recognition particle. It theoretically possesses two small open reading frames (smORFs) and encodes a micropeptide of 7 and 16 amino acids from the 5' side. However, it is unknown if those smORFs are translated. Therefore, we constructed a plasmid vector expressing RN7SL1-GFP, in which the former sequence was replaced by AcGFP1, and examined whether GFP is expressed. In 293T cells electroporated or lipofected with the RN7SL1-GFP-expressing plasmid, GFP fluorescence was detected under the microscope in both cases, although there were a few GFP-positive cells. GFP-positive and GFP-negative cells were separated by FACS and gene expression of each sample was confirmed by RNA-seq. As a result, GFP-positive cells showed activation of cancer-related pathways compared to GFP-negative cells. These results suggest that RN7SL1 smORFs may be translated under special conditions.

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Human hepatic micropeptide LC-MS/MS

Project description:Considering the large number of sORFs in eukaryotic cells, micropeptides might be a long-neglected potential resource pool. However, due to the methodological limitations of micropeptide identification, tumor-related micropeptides have not been comprehensively identified, and their functional mechanisms in tumor progression remained largely uncharacterized. In the present study, we systematically identified micropeptides within HCC tissue samples based on mass spectrometry (MS) detection. By producing a high-coverage micropeptide sequencing reference library and devising an alternative ultrafiltration-concentrated tandem mass spectrometry assay, 1532 unannotated micropeptides (with no overlap with previously annotated CDSs) were successfully identified. It turned out that 81.5% of these micropeptides derived from novel ORFs and 70% of them used non-ATG start codons. Noteworthy, 5.3% of micropeptides were derived from lncRNAs, which are previously considered incapable of protein-coding. Our systematic screening of liver cancer-related micropeptides and in-depth exploration of their functions will improve our understanding of HCC pathogenesis and molecular targeted therapy.

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TGF-β/Nodal signaling boosts glucose uptake during mesendoderm differentiation by inducing a transmembrane micropeptide

Project description:TGF-β signaling is a central regulator of early development in metazoans, yet our understanding of the scope of TGF-β signaling’s downstream targets and associated physiological mechanisms in specifying developmentally appropriate cell fates is far from complete. Here, we found that a highly conserved, primitive-streak-specific micropeptide is a direct target of TGF-b/Nodal signaling. This transmembrane micropeptide (NEMEP) is essential for mesendoderm differentiation. Depletion of NEMEP impaired mesendoderm differentiation and caused a significant decrease in glucose uptake, while TGF-β signaling enhances glucose uptake in a NEMEP-dependent manner. Biochemically, we show that NEMEP promotes glucose uptake through its interactions with GLUT1/3. Thus, beyond expanding the scope of known TGF-β signaling targets in early development and showing that this target micropeptide augments the glucose uptake function of major glucose transporters during mesendoderm differentiation, our study provides a clear example for the direct functional impact of altered metabolism on cell fate determination in early embryogenesis.

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The Clinical Significance of CRNDE Gene Methylation, Polymorphisms, and CRNDEP Micropeptide Expression in Ovarian Tumors

Project description:Abstract: CRNDE is an oncogene expressed as a long non-coding RNA. However, our team previously reported that the CRNDE gene also encodes a micropeptide, CRNDEP. The amino acid sequence of CRNDEP has recently been revealed by other researchers, too. This study aimed to investigate genetic alterations within the CRNDEP-coding region of the CRNDE gene, methylation profiling of this gene, and CRNDEP expression analysis. All investigations were performed on clinical material from patients with ovarian tumors of diverse aggressiveness. We found that CRNDEP levels were significantly elevated in highly aggressive tumors compared to benign neoplasms. Consistently, a high level of this micropeptide was a negative, independent, prognostic, and predictive factor in high-grade ovarian cancer (hgOvCa) patients. The cancer-promoting role of CRNDE(P), shown in our recent study, was also supported by genetic and epigenetic results obtained herein, revealing no CRNDEP-disrupting mutations in any clinical sample. Moreover, in borderline ovarian tumors (BOTS), but not in ovarian cancers, the presence of a single nucleotide polymorphism in CRNDE, rs115515594, significantly increased the risk of recurrence. Consistently, in BOTS only, the same genetic variant was highly overrepresented compared to healthy individuals. We also discovered that hypomethylation of CRNDE is associated with increased aggressiveness of ovarian tumors. Accordingly, hypomethylation of this gene’s promoter/first exon correlated with hgOvCa resistance to chemotherapy, but only in specimens with accumulation of the TP53 tumor suppressor protein. Taken together, these results contribute to a better understanding of the role of CRNDE(P) in tumorigenesis and potentially may lead to improvements in screening, diagnosis, and treatment of ovarian neoplasms.

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A Multi-Faceted Analysis Showing CRNDE Transcripts and a Recently Confirmed Micropeptide as Important Players in Ovarian Carcinogenesis (identification of the CRNDEP micropeptide by immunoprecipitation followed by MS in one OvCa sample)

Project description:Abstract: CRNDE is considered an oncogene expressed as long non-coding RNA. Our previous paper is the only one reporting CRNDE as a micropeptide-coding gene. The amino acid sequence of this micropeptide (CRNDEP) has recently been confirmed by other researchers. This study aimed at the mass spectrometry(MS)-based validation of the CRNDEP sequence and investigation of how the differential expression of CRNDE(P) influences the metabolism and chemoresistance of ovarian cancer (OvCa) cells. We also assessed cellular localization changes of CRNDEP, looked for its protein partners, and bioinformatically evaluated its RNA-binding capacities. Herein, we detected most of the CRNDEP sequence by MS. Moreover, our results corroborated the oncogenic role of CRNDE, portraying it as the gene impacting carcinogenesis at the stages of DNA transcription and replication, affecting the RNA metabolism, and stimulating the cell cycle progression and proliferation, with CRNDEP being detected in the centrosomes of dividing cells. We also showed that CRNDEP is located in nucleoli, and revealed interactions of this micropeptide with p54, an RNA helicase. Besides, we proved the high CRNDE(P) expression to increase the resistance of OvCa cells to treatment with microtubule-targeted cytostatics. Furthermore, altered CRNDE(P) expression affected the activity of the microtubular cytoskeleton and the formation of focal adhesion plaques. Finally, according to our in silico analyses, CRNDEP is likely capable of RNA binding. All these results contribute to a better understanding of the CRNDE(P) role in OvCa biology, which may potentially improve screening, diagnosis, and treatment of this disease.

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