Detection and quantification of RNA decay intermediates using XRN1-resistant reporter transcripts.

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Cellular RNA degradation is crucial for regulating gene expression and eliminating aberrant transcripts.
• Studying RNA degradation intermediates has been challenging due to their inherent instability.
• Existing methods lack the ability to visualize spatiotemporal dynamics of mRNA decay.

Purpose of the Study:

• To develop and detail a novel method for visualizing and quantifying RNA degradation intermediates.
• To characterize the spatiotemporal dynamics of messenger RNA (mRNA) decay in unperturbed cells.
• To enable the study of RNA decay directionality and products.

Main Methods:

• Development of XRN1-resistant RNA sequences (xrRNAs) to protect mRNA from degradation.
• Utilizing xrRNAs in reporter constructs for transfection and cell line generation.
• Employing Northern blotting, quantitative PCR, and single-molecule fluorescence microscopy for detection and quantification.
• Implementing high-throughput image analysis pipelines for spot co-localization and normalization.

Main Results:

• The xrRNA method allows for the visualization of mRNA decay intermediates.
• This approach enables the tracking of degradation products and determination of decay directionality.
• Detailed protocols for reporter design, sample preparation, imaging, and data analysis are provided.
• Analysis software is available to facilitate the detection and quantification of RNA decay intermediates.

Conclusions:

• The xrRNA method overcomes previous limitations in studying RNA degradation dynamics.
• This technique provides unprecedented insights into the spatiotemporal aspects of mRNA decay.
• The developed procedures and software empower researchers to investigate RNA decay mechanisms more effectively.