Abstract
SummaryThe U5 small nuclear ribonucleoprotein particle (snRNP) helicase Brr2 disrupts the U4/U6 small nuclear RNA (snRNA) duplex and allows U6 snRNA to engage in an intricate RNA network at the active center of the spliceosome. Here, we present the structure of yeast Brr2 in complex with the Jab1/MPN domain of Prp8, which stimulates Brr2 activity. Contrary to previous reports, our crystal structure and mutagenesis data show that the Jab1/MPN domain binds exclusively to the N-terminal helicase cassette. The residues in the Jab1/MPN domain, whose mutations in human Prp8 cause the degenerative eye disease retinitis pigmentosa, are found at or near the interface with Brr2, clarifying its molecular pathology. In the cytoplasm, Prp8 forms a precursor complex with U5 snRNA, seven Sm proteins, Snu114, and Aar2, but after nuclear import, Brr2 replaces Aar2 to form mature U5 snRNP. Our structure explains why Aar2 and Brr2 are mutually exclusive and provides important insights into the assembly of U5 snRNP.
Highlights
The spliceosome is a dynamic molecular machine that catalyzes the excision of introns from premessenger RNA and ligation of exons in a two-step trans-esterification reaction
After lariat introns and mature mRNA are released from the spliceosome, U2, U5, and U6 small nuclear ribonucleoprotein particle (snRNP) are recycled for the round of splicing, and Brr2 plays a role in this process (Small et al, 2006)
Experimental phases were obtained to 4.5 Aresolution by single isomorphous replacement anomalous scattering (SIRAS) using a K2OsO4 derivative and extended to 3.1 Aof the native amplitudes by density modification with inclusion of the molecular replacement solutions of the Jab1/MPN domain (Pena et al, 2007) and the second Sec63 unit of Brr2 as partial models (Pena et al, 2009; Zhang et al, 2009; see Experimental Procedures and Figures S1A and S1B available online)
Summary
The spliceosome is a dynamic molecular machine that catalyzes the excision of introns from premessenger RNA (pre-mRNA) and ligation of exons in a two-step trans-esterification reaction. It comprises five RNA-protein subunits, namely U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs), and various non-snRNP factors, which assemble on pre-mRNA substrates in a highly ordered manner (Wahl et al, 2009). Three U5 snRNP proteins, the DExD/H-box family helicase Brr, the EF2-like GTPase Snu114, and Prp, play crucial roles in the activation of the spliceosome and the formation of the catalytic core for the two trans-esterification reactions (Wahl et al, 2009). After lariat introns and mature mRNA are released from the spliceosome, U2, U5, and U6 snRNPs are recycled for the round of splicing, and Brr plays a role in this process (Small et al, 2006)
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