Structure-function analysis of microRNA 3'-end trimming by Nibbler

Area of Science:

• Molecular Biology
• Structural Biology
• RNA Biology

Background:

• Nibbler (Nbr) is a 3'-to-5' exoribonuclease crucial for microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis.
• Understanding Nbr's domain functions is key to elucidating small RNA maturation pathways.

Purpose of the Study:

• To determine the structural and functional roles of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming.
• To investigate the molecular mechanisms underlying Nbr's substrate recognition and catalytic activity.

Main Methods:

• X-ray crystallography to solve apo-state structures of Nbr's NTD core and EXO domains.
• Structure-guided functional assays in Drosophila S2 cells.
• Biochemical assays to characterize Nbr's exoribonuclease activity.

Main Results:

• The Nbr NTD core features a HEAT-like scaffold with basic patches for double-strand RNA (dsRNA) binding, essential for miRNA trimming.
• The Nbr EXO domain possesses Mn2+-dependent, single-strand RNA (ssRNA)-specific 3'-to-5' exoribonuclease activity.
• 2'-O-methylation of 3'-terminal uridine likely protects small RNAs from Nbr trimming due to steric hindrance.

Conclusions:

• Nbr utilizes its NTD as a platform for recruiting small RNA substrates.
• The EXO domain catalyzes the essential exonucleolytic trimming activity for miRNA maturation.
• Nbr's dual-domain mechanism highlights coordinated action in small RNA processing.