PDBsum entry 3e66

Splicing

PDB id: 3e66

Contents

Protein chains

255 a.a.

Waters ×247

References listed in PDB file

Key reference

Title

Crystal structure of the beta-Finger domain of prp8 reveals analogy to ribosomal proteins.

Authors

K.Yang, L.Zhang, T.Xu, A.Heroux, R.Zhao.

Ref.

Proc Natl Acad Sci U S A, 2008, 105, 13817-13822. [DOI no: 10.1073/pnas.0805960105]

PubMed id

18779563

Abstract

Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a beta-hairpin finger protruding out of the protein (hence, this domain will be referred to as the beta-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the beta-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the beta-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its beta-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the beta-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome.

Figure 2.
Structure of the β-finger domain of yPrp8. (A) The β-finger domain structure is colored in a rainbow spectrum from the N terminus to the C terminus, with secondary structures labeled. (B) The most striking feature of the β-finger domain is a protruding β-finger which adopts different conformations in the two molecules (yellow and cyan) in the asymmetric unit of the crystal. (C) The N-terminal α/β subdomain of the β-finger domain (cyan) is topologically similar to the RNase H fold as exemplified by the RNase H domain in the C-terminal of UvrC (yellow) (PDB ID code 2NRR). The extra α-helix and β-strand that are present in the β-finger domain but not in UvrC are labeled with arrows. Residues in Prp8 that correspond to the DDE active sites in RNase H are shown in purple. (D) The β-finger domain contains first-step alleles (purple), second-step alleles (red), and U4-cs1 suppressors (black). Brown designates the residue that confers both the first-step allele and U4-cs-1 suppressor phenotypes. (E) Examples of two ribosomal proteins (S10 and L22) with β-finger extensions.

Figure 4.
Copper-resistance and U4-cs1 suppression analyses of various β-finger mutations. (A) All β-finger mutations grow similarly to the WT at 30°C, 18°C, and 37°C. Only one concentration point in the serial dilution is shown. (B) Copper-resistance assay indicates that V1860D, T1865K, A1871E, and T1872E grow worse than the WT in both the BSG and UuG reporters at 0.05 mM Cu^++ concentration, characteristic of first-step alleles. Mutant H1863E grows better than the WT in both the BSG and UuG reporters at 0.2 mM Cu^++ concentration, characteristic of second-step alleles. V1862D behaves similarly to the WT and does not demonstrate a clear first- or second-step allele phenotype. Known first-step allele R1753K and second-step allele prp8–162 (V1870N) are used as positive controls and labeled with +. (C) Primer extension experiment indicates that V1860D, T1865K, A1871E, and T1872E are first-step alleles, which demonstrate increased lariat intermediate, reduced mRNA product, and reduced second-step efficiency compared with the WT. H1863E is a second-step allele, which demonstrates decreased lariat intermediate, increased mRNA product, and increased second-step efficiency compared to the WT. V1862D is neither a clear first- nor second-step allele. R1753K and prp8–162 (V1870N) are used as positive controls for first and second-step alleles, and the corresponding lanes are labeled with +. pBR322 DNA digested with MspI is used as a molecular weight marker. (D) V1860D (positive control, designated with +) but no other β-finger mutants tested suppress the U4-cs1 phenotype at 18°C. Only one concentration point in the serial dilution is shown.