RNAi Screening to Identify Factors That Control Circular RNA Localization.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Circular RNAs (circRNAs) are formed by noncanonical splicing of eukaryotic protein-coding genes.
• circRNAs are resistant to exonuclease degradation, potentially accumulating at higher levels than linear mRNAs.
• The functions of most circRNAs remain largely unknown, though proposed roles include microRNA sponging, protein translation, and immune response modulation.

Purpose of the Study:

• To describe a method for identifying regulators of circular RNA localization in Drosophila cells.
• To investigate the mechanisms of nuclear export and subcellular localization of circRNAs.
• To discover novel factors that control circRNA localization.

Main Methods:

• RNA interference (RNAi) screening was employed to deplete specific factors.
• Biochemical fractionation was used to separate nuclear and cytoplasmic cellular components.
• Quantitative reverse transcription PCR (RT-qPCR) with primers spanning backsplicing junctions quantified circRNA levels in different compartments.

Main Results:

• The study presents a combined RNAi screening, subcellular fractionation, and RT-qPCR approach.
• This method allows for the quantification of circRNAs in nuclear and cytoplasmic fractions.
• The approach is effective for identifying regulators of circRNA localization.

Conclusions:

• The described methodology provides a robust framework for studying circRNA nuclear export and localization.
• This approach can identify novel regulatory factors influencing circRNA subcellular distribution.
• Understanding circRNA localization mechanisms is crucial for elucidating their diverse functions.