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Published on: June 23, 2022
MicroRNA-223 Regulates Septin-2 and Septin-6 in Stored Platelets
Maitreyi Chattopadhyay1, Neetu Dahiya1, Chintamani Atreya1
1Laboratory of Cellular Hematology, Division of Blood Components and Devices, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Building 71, Room 4236, Silver Spring, MD 20993-0002, United States.
Background:
Septins have been identified to play important roles in platelets, but their regulation in platelets is unknown. Human platelet being an enucleated and terminally differentiated cell, mRNA downregulation by miRs is one of the posttranscriptional mechanisms operative in platelets.
Objective:
Since platelets are known to have miR-223 in abundance, the objective of this study is to test whether a) platelet septins have miR-223 interacting target sites in their mRNA 3'UTRs, b) septin mRNAs and miR-223 form complexes with Argonaute 2 (AGO2) protein in platelets, which is the catalytic component of an RNA Induced Silencing Complex (RISC), c) a reporter gene with septin mRNA 3' untranslated region (UTR) is subjected to downregulation by miR-223 and d) anti-miR-223 can suppress miR-223 activity and enhance septin-2 expression in platelets.
Method:
Bioinformatics tools were used to screen mRNA 3'UTRs of septin-2 and septin-6 for miR- 223 target sites. Subsequently, platelet extracts were immunoprecipitated by AGO2 antibodies to identify that the two septin mRNAs and miR-223 were in complex with AGO2. A luciferase reporter chimeric- gene expression system was utilized to monitor miR-223 mediated downregulation luciferase gene containing the 3'UTR of either septin-2 or septin-6. Further, anti-miR-223 was utilized in platelets to directly demonstrate the role of miR-223 on the expression of septin-2.
Results:
Our results demonstrate that in stored platelets a) septine-2 and septin-6 mRNAs have miR- 223 target sites, b) septin-2 and septin-6 are in complex with Ago-2, c) in luciferase reporter gene system, the interaction of miR-223 with 3' UTRs of septin-2 and septin-6 leads to downregulation of luciferase expression and d) anti-miR-223 downregulated miR-223 activity and thereby the expression of septin-2 is upregulated.
Conclusion:
The results demonstrate that like in nucleated cells, enucleated platelets also have miRbased mechanisms for the regulation of their septins.
Insights
Platelet septins are regulated by microRNA-223 (miR-223) through mRNA targeting, similar to nucleated cells. This study confirms miR-223 downregulates septin expression in enucleated platelets.
Area of Science:
- Molecular Biology
- Cell Biology
- Hematology
Background:
- Septins are crucial proteins in platelet function.
- Platelet regulation mechanisms, especially post-transcriptional, are not fully understood.
- MicroRNAs (miRs) are key regulators of gene expression in enucleated cells like platelets.
Purpose of the Study:
- To investigate the role of miR-223 in regulating septin expression in human platelets.
- To identify miR-223 target sites in septin mRNAs.
- To determine if miR-223 interacts with Argonaute 2 (AGO2) and affects septin mRNA levels.
Main Methods:
- Bioinformatic analysis of septin mRNA 3'UTRs for miR-223 binding sites.
- Immunoprecipitation of Argonaute 2 (AGO2) to detect septin mRNA and miR-223 complexes.
- Luciferase reporter assays to assess miR-223-mediated gene silencing.
- Inhibition of miR-223 using anti-miR-223 in platelets to observe effects on septin-2 expression.
Main Results:
- Septin-2 and septin-6 mRNAs possess target sites for miR-223.
- Septin-2 and septin-6 mRNAs, along with miR-223, were found to be complexed with AGO2 in platelets.
- miR-223 binding to septin 3'UTRs significantly downregulated reporter gene expression.
- Blocking miR-223 activity with anti-miR-223 led to increased septin-2 expression in platelets.
Conclusions:
- Enucleated platelets utilize miR-based mechanisms for septin gene regulation, mirroring processes in nucleated cells.
- miR-223 is a direct regulator of septin expression in platelets.
- This finding expands our understanding of post-transcriptional regulation in anucleated cells.
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