The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.
The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, ... followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.
Mesh Terms:
Adenosine Triphosphatases, Chaetomium, Crystallization, Humans, Models, Molecular, Protein Binding, Protein Folding, Protein Splicing, Protein Structure, Quaternary, Protein Structure, Tertiary, RNA Helicases, RNA-Binding Proteins, Ribonucleoprotein, U4-U6 Small Nuclear, Ribonucleoprotein, U5 Small Nuclear, Ribonucleoproteins, Small Nuclear, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spliceosomes
Adenosine Triphosphatases, Chaetomium, Crystallization, Humans, Models, Molecular, Protein Binding, Protein Folding, Protein Splicing, Protein Structure, Quaternary, Protein Structure, Tertiary, RNA Helicases, RNA-Binding Proteins, Ribonucleoprotein, U4-U6 Small Nuclear, Ribonucleoprotein, U5 Small Nuclear, Ribonucleoproteins, Small Nuclear, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spliceosomes
Genes Dev.
Date: Dec. 15, 2015
PubMed ID: 26637280
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