The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2
Agnieszka Rybak1, Heiko Fuchs, Kamyar Hadian
1Center for Anatomy, Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Abstract:
The let-7 miRNA and its target gene Lin-28 interact in a regulatory circuit controlling pluripotency. We investigated an additional let-7 target, mLin41 (mouse homologue of lin-41), as a potential contributor to this circuit. We demonstrate the presence of mLin41 protein in several stem cell niches, including the embryonic ectoderm, epidermis and male germ line. mLin41 colocalized to cytoplasmic foci with P-body markers and the miRNA pathway proteins Ago2, Mov10 and Tnrc6b. In co-precipitation assays, mLin41 interacted with Dicer and the Argonaute proteins Ago1, Ago2 and Ago4. Moreover, we show that mLin41 acts as an E3 ubiquitin ligase in an auto-ubiquitylation assay and that mLin41 mediates ubiquitylation of Ago2 in vitro and in vivo. Overexpression and depletion of mLin41 led to inverse changes in the level of Ago2 protein, implicating mLin41 in the regulation of Ago2 turnover. mLin41 interfered with silencing of target mRNAs for let-7 and miR-124, at least in part by antagonizing Ago2. Furthermore, mLin41 cooperated with the pluripotency factor Lin-28 in suppressing let-7 activity, revealing a dual control mechanism regulating let-7 in stem cells.
Insights
The study reveals mLin41 protein regulates stem cell pluripotency by controlling Ago2 protein levels and microRNA activity. mLin41 works with Lin-28 to fine-tune let-7 microRNA function in stem cells.
Area of Science:
- Stem cell biology
- Molecular biology
- Epigenetics
Background:
- The let-7 microRNA (miRNA) and Lin-28 gene form a regulatory circuit critical for maintaining stem cell pluripotency.
- The mouse homologue of lin-41, mLin41, is a potential component of this pluripotency regulatory circuit.
Purpose of the Study:
- To investigate the role of mLin41 protein in stem cell niches and its interaction with the let-7/Lin-28 regulatory circuit.
- To elucidate the molecular mechanisms by which mLin41 influences miRNA pathway components and stem cell pluripotency.
Main Methods:
- Immunofluorescence to detect mLin41 protein localization in stem cell niches.
- Co-precipitation assays to identify mLin41 interacting partners within the miRNA pathway.
- In vitro and in vivo ubiquitylation assays to assess mLin41's E3 ubiquitin ligase activity.
- Overexpression and depletion studies to evaluate mLin41's impact on Ago2 protein levels and miRNA target silencing.
Main Results:
- mLin41 protein is present in key stem cell niches and localizes to cytoplasmic foci with miRNA pathway proteins.
- mLin41 interacts with Dicer and Argonaute proteins (Ago1, Ago2, Ago4) and functions as an E3 ubiquitin ligase, mediating Ago2 ubiquitylation.
- mLin41 regulates Ago2 protein turnover and antagonizes Ago2 function, thereby interfering with the silencing of let-7 and miR-124 target mRNAs.
- mLin41 cooperates with Lin-28 to suppress let-7 activity, indicating a dual control mechanism for let-7 regulation in stem cells.
Conclusions:
- mLin41 is a novel component of the let-7/Lin-28 regulatory circuit, playing a significant role in stem cell pluripotency.
- mLin41 regulates stem cell pluripotency through its E3 ubiquitin ligase activity, controlling Ago2 protein stability and miRNA-mediated gene silencing.
- The findings reveal a sophisticated dual control mechanism involving mLin41 and Lin-28 in regulating let-7 activity within stem cells.
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