Structural and functional analysis of essential pre-mRNA splicing factor Prp19p

Abstract

U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome. Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability. Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil. Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p. Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils. These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC.

FIG. 1.

(A) Schematic model of pre-mRNA splicing. (B) Organization of characterized domains within Prp19p. The dark shaded portion represents the U-box domain, while the light shaded portion represents the WD40 repeats. The predicted coiled-coil domain is labeled C-C.

FIG. 2.

Prp19p, but not Cef1p, self-associates in vivo. (A) S. cerevisiae Prp19p-HA3 coimmunoprecipitates Prp19p-myc13 in vivo. Anti-HA (upper panel) and anti-myc (lower panel) immunoblots of immunoprecipitates (IP) from prp19-HA3, prp19-myc13, and prp19-HA3 prp19-myc13 strains are shown. Immunoprecipitations were performed with anti-HA antibodies or anti-myc antibodies. (B) S. pombe Cwf8p-HA3 and Cwf8p-myc13 associate in vivo. Anti-HA (upper panel) and anti-myc (lower panel) immunoblots of IP from cwf8-HA3, cwf8-myc13, and cwf8-HA3 cwf8-myc13 strains are shown. Immunoprecipitations were performed with anti-HA antibodies or anti-myc antibodies. An asterisk indicates the position of the immunoglobulin G heavy band. (C) S. cerevisiae Cef1p-HA3 does not associate with Cef1p-myc13 in vivo. Anti-HA (upper panel) and anti-myc (lower panel) immunoblots of IP from cef1-HA3, cef1-myc13, and cef1-HA3 cef1-myc13 strains are shown. Immunoprecipitations were performed with anti-HA antibodies or anti-myc antibodies. (D) S. pombe Cdc5p-HA3 does not coimmunoprecipitate Cdc5p-myc13 in vivo. An anti-myc (upper panel), an anti-Cdc5 (middle panel), and an anti-HA (lower panel) immunoblot of IP from cdc5-HA3, cdc5-myc13, and cdc5-HA3 cdc5-myc13 strains are shown. Immunoprecipitations were performed with preimmune sera (PI), anti-HA antibodies, anti-myc antibodies, or anti-Cdc5p immune sera (cdc5). (E) The Cdc5p-TAP complex contains more than one copy of Cwf8p. Anti-myc and anti-HA immunoblots of a protein lysate prepared from S. pombe wild-type strain (wt) (KGY246) or of an eluate from a tandem affinity purification from the diploid cdc5-TAP/cdc5-TAP cwf8-HA3/cwf8-myc13 strain (KGY4986) (left panels) are shown. Immunoprecipitations (IP) were performed on the TAP eluate from the diploid strain KGY4986 with anti-HA antibodies and anti-myc antibodies and then immunoblotted with anti-myc antibodies (right panel).

FIG. 3.

Mapping Prp19p interactions with itself and Cef1p. (A) PJ69-A was transformed with pGBT9PRP19 1-131 and screened with the two-hybrid library YHL-1. The bar graph shows β-galactosidase activity (represented by relative light units) of the strains containing the bait and prey constructs indicated. (B) The PJ69-4A strain was transformed with pGBT9PRP19 57-144 and pGAD424 carrying PRP19 57-144. The bar graph shows β-galactosidase activity (represented by relative light units) of the strains containing the bait and prey constructs indicated. (C) The PJ69-4A strain was transformed with pGBT9CEF1 427-590 and pGAD424PRP19 74-134, which are shown schematically above the bar graph. The bar graph shows β-galactosidase activity (represented by relative light units) of the strains containing the bait and prey constructs indicated.

FIG. 4.

Continuous size distribution analysis of sedimentation velocity data of His₆-Prp19p and Prp19p 66-141. (A) The calculated s [c(s)] is plotted versus the sedimentation coefficient (s) for His₆-Prp19p. The sedimentation velocity profiles were fitted to a continuous sedimentation distribution. (B) Calculated mass [c(M)] is plotted versus molar mass (in kilodaltons) for His₆-Prp19p. (C) c(s) plotted versus sedimentation coefficients (s) for Prp19p 66-141. The sedimentation velocity profiles were fit to a continuous sedimentation distribution. (D) c(M) plotted versus molar mass (in kilodaltons) for Prp19p 66-141. (E) CD spectrum of Prp19p 66-141.

FIG. 5.

Electron micrograph and representative projection averages of negatively stained His₆-Prp19p. (Upper panel) Typical micrograph area of negatively stained His₆-Prp19p. Bar, 50 nm. (Lower panel) Twelve representative averages obtained by multireference alignment and classification of 9,725 His₆-Prp19p particle images. The averages reveal the variability of the position of the globular domains in relation to the central stalk. Side length of the average images is 40 nm. Number of particles in each projection average: panel 1, 50; panel 2, 51; panel 3, 35; panel 4, 30; panel 5, 35; panel 6, 39; panel 7, 41; panel 8, 22; panel 9, 64; panel 10, 7; panel 11, 46; panel 12, 22.

FIG. 6.

Electron micrograph and representative projection averages of negatively stained His₆-Prp19p 165-503. (Upper panel) Typical micrograph area of negatively stained His₆-Prp19p. Bar, 25 nm. (Lower panel) Twelve representative averages obtained by multireference alignment and classification of 4,507 His₆-Prp19p 165-503 particle images. Side length of the average images is 16.8 nm. Number of particles in each projection average: panel 1, 168; panel 2, 554; panel 3, 185; panel 4, 224; panel 5, 244; panel 6, 409; panel 7, 205; panel 8, 187; panel 9, 193; panel 10, 216; panel 11, 179; panel 12, 197.

FIG. 7.

Mutational analysis of the Prp19p coiled-coil region. (A) Cwf8p/Prp19p is present within the NTC in a 4:1 ratio with Cdc5p/Cef1p. An anti-myc immunoblot of an anti-HA immunoprecipitate (IP) from either a wild-type S. pombe strain (KGY246) or from cwf7-HA3, cdc5-myc13, or cwf8-myc13 (KGY4985). (B) Gel filtration elution profiles of His₆-Prp19p (black line), His₆-Prp19p-W88A (grey line), and His₆-Prp19p-W88P (dashed line). (C) Typical micrograph area of negatively stained His₆-Prp19p-W88A. Bar, 50 nm. (D) Typical micrograph area of negatively stained His₆-Prp19p-W88P. Arrows point to the flexible coiled-coil region of Prp19p that can no longer oligomerize. Bar, 50 nm. (E) Haploid strain containing a temperature-sensitive copy of PRP19 (prp19-1) (KGY1811) was transformed with pRS415 plasmids containing wild-type PRP19 and prp19 coil-coil mutations under the control of the GAL1 promoter. The ability of prp19 mutations to rescue growth of the prp19-1 strain was scored by serial dilutions grown on medium lacking glucose at the permissive temperature (25°C) or at the restrictive temperature (36°C) for 3 days. (F) A prp19::HIS3 ura3-52 leu2-Δ1 haploid strain carrying a URA3-selectable vector expressing wild-type PRP19 was transformed by a standard lithium acetate method with PRP19 cDNA and prp19 mutations under control of the GAL1 promoter in a LEU2-based vector. Ura⁺ Leu⁺ transformants were serially diluted on plates containing 5-fluoroorotic acid and uracil to score the ability of prp19 mutations to rescue growth of the prp19Δ strain.

FIG. 8.

Model for NTC organization. (A) Representative projection average of the Prp19p tetramer. Side length of the average image is 40 nm. (B) Model of protein-protein interactions mapped between Prp19p, Cef1p, Cwc2p, Prp46p, Syf1p, Isy1p, Snt309p, and Ntc20. U, U-box domain in Prp19p; C-C, coiled-coil region in Prp19p. Solid line with arrow, protein-protein interactions detected by in vitro binding assays and far Western analysis (5, 7, 27, 35); dotted lines, protein-protein interactions found by two-hybrid analysis (27). Proteins and domains are not drawn to scale.