Structure of an RNase H domain in Prp8. (A) Ribbon diagram of scPrp8^1827−2092 (left) and human RNase H1 (right; pdb ID 2QKK; Nowotny et al, 2007) in the same orientations. Top panels: the shape of scPrp8^1827−2092 resembles a mitten (inset) with palm, thumb and finger regions. Secondary structure elements and termini are labelled. Equivalent α-helices and β-strands in Prp8 and RNase H are blue and red, respectively; β-hairpin comprising the thumb (strands β1a and β1b)—magenta; η5 encompassing the peptide crosslinked to the 5′SS in human Prp8—cyan; C-terminal domain of Prp8—green. Acidic residues I–IV involved in metal ion binding in RNase H (the asparagine at position III is an aspartate in the wild-type enzyme) as well as D1853 and D1854 in Prp8, which may correspond to carboxylate I, are shown as sticks (carbon—yellow; oxygen—red; nitrogen—blue). Bottom panels: view on the RNase H-like NTD of scPrp8^1827−2092 (left) and hsRNase H1 (right) rotated 90° about the vertical axis as compared with the top panels. The CTD of scPrp8^1827−2092 was removed for clarity. In scPrp8^1827−2092, the C-terminal helix α3 of hsRNase H1 is divided into helix η5 and the perpendicular helix α3. (B) Electrostatic potential mapped to the surface of scPrp8^1827−2092. Blue—positive charge; red—negative charge. The position of the crosslinked peptide is circled (‘XL'). (C) Superimposition of scPrp8^1836−2092 (white/yellow) with two independent structure of scPrp8^1827−2092 (light grey/green and dark grey/red) and with hsPrp8^1755−2014 (black/blue). The thumbs, the peptides that crosslink to the 5′SS in the human system (‘XL peptide') and the C-terminal portions of the fingers are shown in colours. Thumbs and fingers are evidently flexibly hinged to the central palms.

Multiple sequence alignment of Prp8 proteins. Darker background corresponds to higher conservation. Aligned orthologues are H.s.—Homo sapiens; N.c.—Neurospora crassa; Y.l.—Yarrowia lipolytica; C.g.—Candida glabrata; S.c.—Saccharomyces cerevisiae. Icons above/below the alignment indicate secondary structure elements of hsPrp8^1755−2016/scPrp8^1827−2092 as derived from the present crystal structures. Residues that exhibit genetic interactions with the 5′SS, 3′SS and BPS—blue; with u4-cs1—red; with the PPy tract—purple. Residues mutated in this work—yellow. Black triangles—conserved residues around the functional hotspot. The region of Prp8 crosslinked to the 5′SS and the region, in which mutations affect the interaction with Brr2, are boxed (cyan and green, respectively). Green residues in the Brr2-interacting region correspond to changes in the prp8-52 allele.

Functional interactions. (A) Diametrical views on the surface of scPrp8^1836−2092 with a modelled RNA mimicking a bound 5′SS region. The left orientation is identical to that in Figure 1A, top. 5′-exon—brown; 5′SS phosphate—black; intron—beige. The conserved GU at positions +1 and +2 of the intron are labelled. The surface patch corresponding to the peptide, which in hsPrp8 crosslinks to the 5′SS, is encircled. Positions that exhibit genetic interactions with the pre-mRNA or snRNAs are highlighted in colour. Interactions with 5′SS, 3′SS or BPS—blue; interactions with the PPy tract—purple (mutations at positions 1922 and 1946 always occur together with a mutation at 1834); interactions with u4-cs1—red. The region, in which mutations affect the physical interaction with Brr2, is in green. Residues changed in Prp8 mutants are listed. Conserved and invariant residues that lie in the region corresponding to the active site in RNase H enzymes (D1853, D1854, T1855, T1936 and R1937) are in gold. (B) RNA networks around the 5′SS shown on top of an outline of the present Prp8 domain. Before catalytic activation, U4 and U6 are base paired 3′ of the U6 ACAGAGA-box. U1 snRNA and U6 ACAGAGA-box interact at the 5′SS. In U4-cs1 (grey), an AAA sequence of U4 (underlined) is changed to UUG (lower case italics), which can form additional base pairs with U6 that involve the ACAGAGA-box as indicated.

Identification of a novel functional hotspot. (A) Active site of hsRNase H1 in complex with two divalent metal ions (A and B; green spheres) and a DNA–RNA substrate duplex (carbons—light/dark grey; phosphorus—violet; other colours as in Figure 1). The four active site carboxylates (I–IV) are shown as sticks and are colour-coded as before (the asparagine at position III is an aspartate in the wild-type enzyme). Landmark secondary structure elements are labelled. The view is rotated 80° about the vertical axis and 20° about the horizontal axis as compared with the view in Figure 1A, top. (B) Close-up of the region of scPrp8^1827−2092, which is spatially equivalent to the RNase H active site, in the same orientation as in (A). The invariant D1853, D1854, T1855, T1936 and R1937 are shown as sticks and are colour-coded as above. Other conserved residues are labelled. Dashed lines indicate hydrogen bonds and salt bridges. Selected secondary structure elements are labelled. D1853 occupies the same topological and spatial position as carboxylate I of hsRNase H1, the carboxylate of D1854 spatially coincides with the carboxylate II of hsRNase H1. (C) Failure of D1853A and R1937A mutants of scPrp8 to grow on 5-FOA plates. (D) Cell viability assay monitoring the effects of exchanging invariant Prp8 residues D1853, D1854, T1855, T1936 and R1937 as indicated. After selection of clones, the culture and serial dilutions were spotted and grown at the indicated temperatures for 2 days.