RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing

Area of Science:

• Molecular Biology
• RNA Splicing
• Structural Biology

Background:

• The SR-like protein Npl3 is crucial for pre-mRNA splicing.
• The specific RNA sequences recognized by Npl3's RNA Recognition Motifs (RRMs) are unknown.

Purpose of the Study:

• To determine the RNA sequences bound by Npl3's RRMs.
• To elucidate the structural basis of Npl3-RNA interactions.
• To investigate Npl3's role in spliceosome active site formation.

Main Methods:

• Split-integrated Crystal of RNA Capture (iCRAC) in yeast.
• High-resolution Nuclear Magnetic Resonance (NMR) spectroscopy.
• Structure-guided functional assays.

Main Results:

• Npl3's RRM2 binds a 5´-GNGG-3´ motif with a mille-feuille topology.
• Npl3's RRM1 binds a CC-dinucleotide upstream of the GNGG motif.
• Npl3 melts U2 snRNA stem-loop I, promoting U2/U6 duplex formation.

Conclusions:

• Npl3 recognizes specific RNA motifs via its RRMs.
• Npl3 functions as an RNA chaperone in spliceosome assembly.
• Npl3's RNA chaperoning activity is essential for spliceosome catalytic center formation.