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Published on: April 11, 2022
Interdependence of Pasha and Drosha for localization and function of the Microprocessor in C. elegans
Thiago L Knittel1, Brooke E Montgomery1, Kailee J Reed1,2
1Department of Biology, Colorado State University, Fort Collins, CO, USA.
Abstract:
Primary microRNA (pri-miRNA) transcripts are processed by the Microprocessor, containing the ribonuclease Drosha and its RNA-binding partner DGCR8/Pasha. In a forward genetic screen utilizing a fluorescence-based sensor that monitors pri-miRNA processing in live Caenorhabditis elegans, we identify a mutation in the conserved G179 residue adjacent to the namesake W180 of Pasha's WW domain that disrupts pri-miRNA processing. We show that both the G179 and W180 residues are required for Pasha dimerization and Microprocessor assembly. The WW domain also facilitates nuclear localization of Pasha, likely through its role in Microprocessor assembly, which in turn promotes nuclear enrichment of Drosha. Furthermore, depletion of Pasha mislocalizes Drosha to the cytoplasm, and vice versa, while deletion of Pasha's N-terminus causes both proteins to accumulate in nucleoli. Our results reveal a mutual dependency between Pasha and Drosha for their localization in C. elegans and highlight the role of Pasha's WW domain in maintaining Microprocessor integrity.
Insights
A mutation in Pasha’s WW domain disrupts microRNA processing in C. elegans. This WW domain is crucial for Pasha dimerization, Microprocessor assembly, and the nuclear localization of both Pasha and Drosha.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Primary microRNA (pri-miRNA) processing is essential for gene regulation.
- The Microprocessor complex, comprising Drosha and DGCR8/Pasha, mediates pri-miRNA processing.
- Understanding the regulation of Microprocessor assembly and function is critical.
Purpose of the Study:
- To identify genetic factors affecting pri-miRNA processing in Caenorhabditis elegans.
- To elucidate the role of Pasha's WW domain in Microprocessor function and protein localization.
Main Methods:
- Forward genetic screen using a fluorescence-based sensor for pri-miRNA processing.
- Site-directed mutagenesis to investigate specific residues (G179, W180) in Pasha's WW domain.
- Analysis of protein localization (nuclear, cytoplasmic, nucleolar) of Drosha and Pasha upon genetic manipulation.
Main Results:
- A mutation in Pasha's WW domain (G179 residue) impairs pri-miRNA processing.
- Both G179 and W180 residues are essential for Pasha dimerization and Microprocessor assembly.
- Pasha's WW domain mediates nuclear localization of Pasha and promotes Drosha nuclear enrichment.
- Pasha and Drosha exhibit mutual dependency for correct subcellular localization.
Conclusions:
- Pasha's WW domain is critical for maintaining Microprocessor integrity and function.
- Proper assembly and localization of the Microprocessor complex are interdependent.
- This study reveals key insights into the regulation of microRNA biogenesis in C. elegans.
