Functional Atlas of Primary miRNA Maturation by the Microprocessor

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Primary microRNAs (miRNAs) are precursor molecules essential for gene expression regulation in humans.
• The Microprocessor complex (Drosha/DGCR8) processes primary miRNAs into mature miRNAs through hairpin structure cleavage.
• Key features distinguishing primary miRNA hairpins from other non-coding RNAs remain incompletely understood.

Purpose of the Study:

• To investigate the molecular features governing primary miRNA recognition and processing by the Microprocessor complex.
• To develop a high-throughput assay for functional analysis of primary miRNA substrates and sequence variants.
• To elucidate the sequence-structure relationship critical for efficient miRNA biogenesis.

Main Methods:

• Development and application of Dro-seq (Drosha sequencing), a massively parallel functional assay.
• Testing of numerous known primary miRNA substrates and thousands of single-nucleotide variants.
• Deep mutagenesis experiments to probe sequence-structure requirements for processing.

Main Results:

• Identification of Shannon entropy as an important feature of primary miRNA structural ensembles for processing.
• Discovery that specific apical loop uracil (U) bases are critical for efficient processing, likely via DGCR8 recognition.
• Characterization of novel sequence-structure determinants for Microprocessor complex activity.

Conclusions:

• Shannon entropy and apical loop U bases represent key features influencing primary miRNA processing.
• The findings provide a deeper understanding of the substrate specificity of the Microprocessor complex.
• This work advances knowledge of miRNA biogenesis and RNA sequence-structure relationships.