Identification of human pathways acting on nuclear non-coding RNAs using the Mirror forward genetic approach

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Nuclear non-coding RNA pathways are crucial in disease but difficult to study using forward genetics.
• Existing methods struggle to investigate strictly nuclear RNA processing and degradation pathways.

Purpose of the Study:

• To develop a novel single-cell genetic approach for discovering nuclear non-coding RNA pathways.
• To identify components of RNA processing and degradation complexes.
• To uncover novel roles for proteins, such as DDX59, in nuclear RNA metabolism and human diseases.

Main Methods:

• Development of a single-cell "Mirror" approach to visualize nuclear RNA pathway activity via cytoplasmic fluorescence.
• Application of the Mirror method to study MALAT1 maturation and degradation pathways, including the Element for Nuclear Expression (ENE).
• Genetic knockout of DDX59 to assess its function in RNA stability and processing.

Main Results:

• The Mirror method successfully identified components of Ribonuclease P, the RNA Exosome (including DIS3, EXOSC10, C1D), and the Nuclear Exosome Targeting (NEXT) complex.
• DEAD-box helicase DDX59 was identified and found to be associated with Oral-Facial-Digital syndrome (OFD).
• DDX59 knockout led to stabilization of MALAT1, increased 3'-extended small nuclear RNAs, and significant minor intron retention, implicating DDX59 in OFD pathogenesis.

Conclusions:

• The Mirror approach is effective for discovering pathways acting on strictly nuclear non-coding RNAs, including essential and indirectly acting components.
• This method can reveal unexpected connections between nuclear RNA metabolism and human genetic disorders like OFD.
• DDX59 plays a significant role in nuclear RNA processing, including intron splicing and small RNA maturation, and its dysfunction may underlie OFD.