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Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

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Microprocessor dynamics and interactions at endogenous imprinted C19MC microRNA genes.

Clément Bellemer¹, Marie-Line Bortolin-Cavaillé, Ute Schmidt

¹Laboratoire de Biologie Moléculaire Eucaryote (LBME), Université Paul Sabatier (UPS), Université de Toulouse, 31000 Toulouse, France.

Journal of Cell Science

|March 7, 2012

Summary

This study visualizes primary microRNA (pri-miRNA) processing in living cells, revealing how the DGCR8-Drosha Microprocessor complex interacts with pri-miRNAs at their genes. It shows Drosha dissociates while DGCR8 remains bound after processing.

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Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

Published on: September 28, 2011

Area of Science:

Molecular Biology
Cell Biology
Genetics

Background:

Nuclear primary microRNA (pri-miRNA) processing by the DGCR8-Drosha Microprocessor complex is crucial but spatially uncharacterized in mammalian nuclei.
Understanding the intranuclear organization of microRNA biogenesis is essential for comprehending gene regulation.

Purpose of the Study:

To visualize the intranuclear distribution and dynamics of endogenously-expressed pri-miRNAs and the Microprocessor complex in living cells.
To investigate the spatial organization of pri-miRNA processing at the human chromosome 19 microRNA cluster (C19MC).

Main Methods:

Live-cell imaging of untagged, endogenously-expressed pri-miRNAs at the C19MC locus.
Single-molecule imaging to track pri-miRNA and Microprocessor complex dynamics within the nucleus.

Main Results:

A significant portion of the Microprocessor complex localizes to nascent C19MC pri-miRNAs near their transcription sites.
DGCR8 and Drosha are recruited as a preformed complex to pri-miRNAs but dissociate independently.
Drosha is released post-cleavage, while DGCR8 remains bound to the processed pri-miRNA.

Conclusions:

Microprocessor complex dynamics during pri-miRNA processing involve conformational changes leading to Drosha release and stable DGCR8 binding.
This study provides the first direct visual evidence of spatial organization in mammalian pri-miRNA processing.
Findings offer insights into the regulation of microRNA biogenesis within the nucleus.