scholarly journals A broad role for YBX1 in defining the small non-coding RNA composition of exosomes

2017 ◽  
Author(s):  
Matthew J. Shurtleff ◽  
Jun Yao ◽  
Yidan Qin ◽  
Ryan M. Nottingham ◽  
Morayma Temoche-Diaz ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Hek293t Cell ◽  
Full Length ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Terminal Oligopyrimidine ◽  
Non Coding Rnas

AbstractRNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their co-isolation with contaminants, a lack of knowledge of the mechanisms of RNA sorting into EVs and limitations of conventional RNA-seq methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspannin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature tRNAs and other small non-coding RNAs encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long non-coding, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5’ terminal oligopyrimidine (5’ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which we showed previously is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small non-coding RNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for post-transcriptional modification in the sorting of some RNA species into EVs. The identification of full-length small non-coding RNAs within EVs suggests a role for EVs in the export and possible intercellular functional transfer of abundant cellular transcripts.Statement of SignificanceCells release vesicles containing selectively packaged cargo, including RNA, into the extracellular environment. Prior studies have identified RNA inside extracellular vesicles (EVs) but, due to limitations of conventional sequencing methods, highly structured and post-transcriptionally modified RNA species were not effectively captured. Using an alternative sequencing approach (TGIRT-seq), we found that EVs contain abundant small non-coding RNA species, including full-length tRNAs and Y RNAs. Using a knockout cell line, we obtained evidence that the RNA-binding protein YBX1 plays a role in sorting small non-coding RNAs into a subpopulation of extracellular vesicles termed exosomes. These experiments expand our understanding of EV-RNA composition and provide insights into how RNA is sorted into EVs for export from the cell.

scholarly journals hnRNPA2B1-Mediated Extracellular Vesicles Sorting of miR-122-5p Potentially Promotes Lung Cancer Progression

2021 ◽  
Vol 22 (23) ◽  
pp. 12866
Author(s):  
Chuang Li ◽  
Fang Qin ◽  
Wei Wang ◽  
Yifan Ni ◽  
Mingyu Gao ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Vesicles ◽  
Cancer Progression ◽  
Tumor Metastasis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Small Cell ◽  
Small Cell Lung ◽  
Non Coding Rna

Extracellular vesicles (EVs) released by tumor cells play important roles on the remodeling of the tumor–stromal environment and on promoting tumor metastasis. Our earlier studies revealed that miR-122-5p, a type of small non-coding RNA, was dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs. In this study, we found that miR-122-5p was selectively sorted and secreted into lung cancer EVs through binding to RNA-binding protein hnRNPA2B1. In addition, we found that hnRNPA2B1 interacted with miR-122-5p through the EXO-motif. The delivering of lung cancer EVs-miR-122-5p promoted the migration of liver cells, which may play roles in establishing a pre-metastatic micro-environment and hepatic metastasis of lung cancer. Importantly, our findings revealed the molecular mechanism that RNA-binding protein controls the selective sorting of tumor-derived EV miR-122-5p, which potentially promotes lung cancer progression.

scholarly journals Broad role for YBX1 in defining the small noncoding RNA composition of exosomes

2017 ◽  
Vol 114 (43) ◽  
pp. E8987-E8995 ◽  
Author(s):  
Matthew J. Shurtleff ◽  
Jun Yao ◽  
Yidan Qin ◽  
Ryan M. Nottingham ◽  
Morayma M. Temoche-Diaz ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Binding ◽  
Hek293t Cell ◽  
Full Length ◽  
Protein Coding ◽  
Transfer Rnas ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Small Ncrnas ◽  
Terminal Oligopyrimidine

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5′ terminal oligopyrimidine (5′ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Our results suggest that EVs and exosomes could play a role in the purging and intercellular transfer of excess free RNAs, including full-length tRNAs and other small ncRNAs.

scholarly journals New Insights into the Interplay between Non-Coding RNAs and RNA-Binding Protein HnRNPK in Regulating Cellular Functions

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 62 ◽  
Author(s):  
Yongjie Xu ◽  
Wei Wu ◽  
Qiu Han ◽  
Yaling Wang ◽  
Cencen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Protein ◽  
Genomic Structure ◽  
Rna Binding Protein ◽  
Stem Cell Differentiation ◽  
Cellular Functions ◽  
Non Coding Rnas ◽  
Conserved Gene

The emerging data indicates that non-coding RNAs (ncRNAs) epresent more than the “junk sequences” of the genome. Both miRNAs and long non-coding RNAs (lncRNAs) are involved in fundamental biological processes, and their deregulation may lead to oncogenesis and other diseases. As an important RNA-binding protein (RBP), heterogeneous nuclear ribonucleoprotein K (hnRNPK) is known to regulate gene expression through the RNA-binding domain involved in various pathways, such as transcription, splicing, and translation. HnRNPK is a highly conserved gene that is abundantly expressed in mammalian cells. The interaction of hnRNPK and ncRNAs defines the novel way through which ncRNAs affect the expression of protein-coding genes and form autoregulatory feedback loops. This review summarizes the interactions of hnRNPK and ncRNAs in regulating gene expression at transcriptional and post-transcriptional levels or by changing the genomic structure, highlighting their involvement in carcinogenesis, glucose metabolism, stem cell differentiation, virus infection and other cellular functions. Drawing connections between such discoveries might provide novel targets to control the biological outputs of cells in response to different stimuli.

scholarly journals Long Non-Coding RNA-Ribonucleoprotein Networks in the Post-Transcriptional Control of Gene Expression

2020 ◽  
Vol 6 (3) ◽  
pp. 40
Author(s):  
Paola Briata ◽  
Roberto Gherzi
Keyword(s):  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Huge Number ◽  
Protein Coding ◽  
Control Of Gene Expression ◽  
Non Coding Rna ◽  
Non Coding Rnas ◽  
And Function ◽  
Long Non Coding Rna

Although mammals possess roughly the same number of protein-coding genes as worms, it is evident that the non-coding transcriptome content has become far broader and more sophisticated during evolution. Indeed, the vital regulatory importance of both short and long non-coding RNAs (lncRNAs) has been demonstrated during the last two decades. RNA binding proteins (RBPs) represent approximately 7.5% of all proteins and regulate the fate and function of a huge number of transcripts thus contributing to ensure cellular homeostasis. Transcriptomic and proteomic studies revealed that RBP-based complexes often include lncRNAs. This review will describe examples of how lncRNA-RBP networks can virtually control all the post-transcriptional events in the cell.

scholarly journals Long Non-Coding RNA H19 Promotes Porcine Satellite Cell Differentiation by Interacting with TDP43

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 259
Author(s):  
Jingxuan Li ◽  
Wenjuan Zhao ◽  
Qianqian Li ◽  
Ziying Huang ◽  
Gaoli Shi ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Biological Processes ◽  
Non Coding Rna ◽  
Non Coding Rnas ◽  
Functional Analyses ◽  
Long Non Coding Rna

Long non-coding RNAs (lncRNAs) have been implicated in fundamental and diverse biological processes, including myogenesis. However, the molecular mechanisms involved in this process remain largely unexplored. This study found that H19 affected the differentiation of porcine satellite cells (PSCs) by directly binding to the DNA/RNA-binding protein TDP43. Functional analyses showed that TDP43 knockdown decreased PSC differentiation, whereas TDP43 overexpression exerted opposite effects in vitro. Furthermore, rescue experiments demonstrated that TDP43 can rescue the decrease in PSC differentiation caused by H19 knockdown. Mechanistically, H19 may act as a scaffold to recruit TDP43 to the promoters of MYOD and thereby activate the transcription of MYOD, leading to PSC differentiation. In summary, we elucidate the molecular mechanism by which H19 and TDP43 regulate myogenesis.

Long non-coding RNA LINC-PINT attenuates paclitaxel resistance in triple-negative breast cancer cells via targeting the RNA-binding protein NONO

2020 ◽  
Vol 52 (8) ◽  
pp. 801-809 ◽  
Author(s):  
Jinghua Chen ◽  
Meiqin Zhu ◽  
Liqiu Zou ◽  
Junxian Xia ◽  
Jiacheng Huang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract The treatment of triple-negative breast cancer (TNBC) relies largely on chemotherapies. However, it is frequent that TNBC patients develop resistance to the chemotherapy drugs. Generation of drug-resistant cell lines facilitates the identification of drug resistance. Here, we established two paclitaxel (PTX)-resistant TNBC cancer cell lines using an intermittent and stepwise method and found that long non-coding RNA long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) was significantly decreased in PTX-resistant cancer cells. Ectopic expression of LINC-PINT sensitized both PTX-resistant TNBC and wild-type TNBC to PTX. Moreover, RNA immunoprecipitation showed that LINC-PINT bound to RNA-binding protein NONO. Overexpression of LINC-PINT resulted in the degradation of NONO in a proteasome-dependent manner and vice versa. Knockdown of NONO with siRNA sensitized TNBC to PTX. We further analyzed the expression level of LINC-PINT and NONO in patient samples via online database and found that LINC-PINT and NONO may function antagonistically in all types of breast cancers. Taken together, our data illustrated a tumor suppressor role of LINC-PINT in sensitizing TNBC to chemotherapies via destabilizing NONO.

scholarly journals The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction

2020 ◽  
Author(s):  
Ewan M Smith ◽  
Nour El Houda Benbahouche ◽  
Katherine Morris ◽  
Ania Wilczynska ◽  
Sarah Gillen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Binding ◽  
Binding Protein ◽  
Mammalian Target Of Rapamycin ◽  
Rna Binding Protein ◽  
Mtor Inhibition ◽  
Growth And Survival ◽  
Mrna Targets ◽  
Terminal Oligopyrimidine ◽  
Rna Interaction

Abstract The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5′ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.

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