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Exploring transcriptomic landscapes in red cell populations, in their extracellular vesicles and on single cell level

ABSTRACT: Circulating human red blood cells (RBCs) consist of mature erythrocytes, and immature reticulocytes. As being anucleated cells, RBCs lack typical, abundant transcriptomes but are known to contain low amounts of diverse long transcripts and microRNAs. The exact role and importance of these RNAs is, however, lacking. To study this further, we have explored the RNA content of RBCs as well as extracellular vesicles of RBCs using next generation sequencing (NGS). Furthermore, to understand the dynamics of the RBC transcriptome we performed single cell RNA sequencing on RBCs both from fresh blood and blood unit. Analysis of the single cell transcriptomes revealed that while the majority of the cells don’t have detectable RNA the remaining, approximately 10% of the cells fall into three sub-populations based on their RNA content. Overall decrease in the RNA quantity over the populations is observed. Qualitative changes include the differences in the hemoglobin (Hb) content of the cells, and the changes in the expression of ribosomal and mitochondrial genes. A previously unknown transcript of a long non-coding RNA, MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is the most enriched marker gene for one subpopulation of RBCs suggesting a role for MALAT1 in reticulocyte maturation. Enriched pathways in RBC transcriptome, such as autophagy, reflect the processes required for reticulocyte maturation to erythrocytes. RBC transcriptome is transferred to extracellular vesicles (EVs) suggesting the vesiculation as the mechanism to remove the cellular contents at the final stages of erythrocyte maturation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE108241 | GEO | 2023/04/01

REPOSITORIES: GEO

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Exploring transcriptomic landscapes in red cell populations, in their extracellular vesicles and on single cell level

Project description:Circulating human red blood cells (RBCs) consist of mature erythrocytes, and immature reticulocytes. As being anucleated cells, RBCs lack typical, abundant transcriptomes but are known to contain low amounts of diverse long transcripts and microRNAs. The exact role and importance of these RNAs is, however, lacking. To study this further, we have explored the RNA content of RBCs as well as extracellular vesicles of RBCs using next-generation sequencing (NGS). Furthermore, to understand the dynamics of the RBC transcriptome, we performed single cell RNA sequencing on RBCs both from fresh blood and blood unit. Analysis of the single cell transcriptomes revealed that while the majority of the cells don’t have detectable RNA, the remaining, approximately 10% of the cells fall into three sub-populations based on their RNA content. Overall decrease in the RNA quantity over the populations is observed. Qualitative changes include the differences in the hemoglobin (Hb) content of the cells, and the changes in the expression of ribosomal and mitochondrial genes. A previously unknown transcript of a long non-coding RNA, MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is the most enriched marker gene for one subpopulation of RBCs, suggesting a role for MALAT1 in reticulocyte maturation. Enriched pathways in RBC transcriptome, such as autophagy, reflect the processes required for reticulocyte maturation to erythrocytes. RBC transcriptome is transferred to extracellular vesicles (EVs), suggesting the vesiculation as the mechanism to remove the cellular contents at the final stages of erythrocyte maturation.

2019-12-20 | GSE108378 | GEO

Comparison of the proteome of human adult and cord erythroid cells and changes in the proteome following reticulocyte maturation.

Project description:Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared to adult peripheral blood progenitors, and cord blood banks usually being more representative of national populations than blood donors. Consequently it is important to establish how similar cord RBCs are to adult cells. In this study we used Multiplex TMTs combined with nanoLC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focussed on proteins critical for RBC function. Of these only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 & 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2 and 100-fold following maturation. However, ~5% were at a higher level in RBCs, localised almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that with respect to the proteome there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal not adult haemoglobin.

2016-03-31 | PXD003276 | Pride

Erythrocyte soluble proteome analysis in hemolytic anaemia patients

Project description:We investigated the RBCs of a hemolytic anemia patient using quantitative proteomics. We used stable isotope dimethyl labeling to accurately quantify the RBC proteins. As controls, 1) samples of four healthy subjects were taken to account for normal variation in healthy individuals, and 2) samples of two non-spherocytic hemolytic anemia patients were taken to account for differences in protein levels due to elevated reticulocyte content. We used a combination of strong cation exchange (SCX) chromatography with nanoLC-MS/MS, enabling quantification of RBC proteins.

2016-09-27 | PXD001837 | Pride

microRNA profiling of RBC transcriptome during ex vivo storage

Project description:Mature human red blood cells (RBCs) are terminally differentiated anuclear cells. While initially thought to lack any nucleic acids, human RBCs are found to contain abundant and diverse species of RNA transcripts with functional relevance. Given the absence of novel transcription, RBCs may provide an interesting cellular context to study RNA metabolism over time. One clinically relevant context is the ex vivo storage of RBCs in blood banks for use in blood transfusion. Some studies have indicated that the transfusion of “old” or aged stored RBCs may be associated with adverse outcomes due to various storage changes termed “storage lesions”. However, other studies do not support these effects, and much remains unknown about the relevant changes associated with RBC storage. Here, we employed the NanoString nCounter assay for global miRNA profiling to comprehensively define the miRNA turnover during ex vivo RBC storage. This profiling demonstrates that the abundance of most RBC miRNAs did not change significantly during the 42 days of refrigerated storage, indicating extremely long decay half-lives. Unexpectedly, miR-720, a cleavage product of tRNAThr, increased dramatically in the first two weeks and persisted during storage. Furthermore, we present evidence for a role of angiogenin in tRNA cleavage to generate miR-720 during RBC storage. The dramatic increase in miR-720 may serve as a new characteristic for storage lesion and may be used to monitor transfused RBCs in clinical patients and athletes performing blood doping.

2020-05-26 | GSE114990 | GEO

Phosphoproteomics and Morphology of Red Blood Cells treated by Protein-Tyrosine-Phosphatases Inhibitor

Project description:Phosphorylation of proteins is involved in cell functions, of which cytoskeleton organization. In particular, the phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions. During the storage of RBC in the context of transfusion medicine, cells suffer from storage lesions that affect energy metabolism as well as cell morphology. In the present research work, RBCs were treated (in absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate, OV) to trigger phosphorylation allowing to investigate this effect on RBCs during the storage. Erythrocytes were studied by phosphoproteomics, Western blot analyses and cell morphology by digital holographic microscopy in presence of kinase inhibitors. The OV treatment triggered phosphorylation events, that disappeared during the storage. As a result, 609 phosphoproteins containing 5’298 phosphosites were detected in the membrane extract, of which 43 pY were up- or down-regulated. Following these phosphorylation processes, shape of RBCs shifted from discocytes to spherocytes, and the addition of kinase inhibitors (KIs) inhibited this transition by counteracting the OV treatment. In addition, the different KIs used highlighted potentially two mechanisms involving membrane proteins and playing a role in RBC shape. The capacity of RBCs to maintain their function is central in transfusion medicine and the presented results contribute to a better understanding of RBC biology.

2024-01-26 | PXD039914 | Pride

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases

Project description:Background Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). Methods and Findings Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. Conclusions In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases. Keywords: Sickle cell disease RBC profiling The microRNA of purified erythrocytes from 7 normal and 12 HbSS individuals

2009-04-04 | E-GEOD-11060 | biostudies-arrayexpress

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

Project description:Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein beta-spectrin. Here we show that activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV-located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in beta-spectrin cleavage and RBC rupture. Drug-like inhibitors of SERA6 autoprocessing similarly prevent beta-spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.

2021-03-16 | PXD024084 | Pride

Small non-coding RNA’s profile of sorted plasma extracellular vesicles expressing different cell origin

Project description:Extracellular vesicles play a pivotal role in the intercellular communications influencing various physiological and pathological processes. They carry a range of biomolecular cargoes including small non-coding RNAs which could serve as potential diagnostic biomarkers and therapeutic targets. In the current study we applied Next Generation Sequencing to investigate small RNA profiles of erythrocytes (Rbc), platelets (Plt), leukocytes (CD45cells), two plasma fractions and blood cell-derived extra-cellular vesicles (EVs, such as CD41+, CD45+, CD235a+, CD146+) obtained using high-sensitivity fluorescence activated vesicle sorting from plasma of healthy donors. We analyzed the proportions of various small ncRNAs across samples and identified sample specific profiles of microRNA (miRNA) and transport RNA-derived fragments (tRFs). It was found that the cumulative small ncRNAs profiles of EVs originated from platelets, erythrocytes, and leukocytes, which are considered to be dominant among the vesicles circulating in blood, differ from small ncRNAs profiles of plasma fractions, which represent macrovesicles and exosomes in circulation. The proportion of miRNAs in sorted EVs was significantly lower compared to other samples while the proportion of tRFs was higher. Moreover, all sorted EVs carried mostly cell type non-specific miRNAs. Taking together, the results demonstrate that the combination of high-sensitivity fluorescence activated vesicle sorting with small-RNA sequencing technique is a powerful approach to analyze small ncRNAs profiles in cell specific vesicles.

2023-03-30 | GSE216355 | GEO

20S proteasomes exported by malaria parasite promote its growth

Project description:Mature Red Blood Cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. While growing inside RBCs, Pf are known to secrete multipurpose extracellular vesicles (EVs), yet their influence on this parasite’s essential host cell, the RBC, remains unknown. Here we demonstrate that Pf parasites export within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve host RBCs by remodeling the cytoskeleton network. Furthermore, we identified four novel degradation targets of the exported 20S proteasome, the phosphorylated cytoskelatal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome export mechanism of Pf-iRBCs, which prime naïve RBC by altering membrane stiffness, to facilitate malaria parasite growth.

2021-01-04 | PXD018154 | Pride

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases

2009-04-04 | GSE11060 | GEO

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