Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors

Abstract

Through exhaustive two-hybrid screens using a budding yeast genomic library, and starting with the splicing factor and DEAH-box RNA helicase Prp22p as bait, we identified yeast Prp45p and Prp46p. We show that as well as interacting in two-hybrid screens, Prp45p and Prp46p interact with each other in vitro. We demonstrate that Prp45p and Prp46p are spliceosome associated throughout the splicing process and both are essential for pre-mRNA splicing. Under nonsplicing conditions they also associate in coprecipitation assays with low levels of the U2, U5, and U6 snRNAs that may indicate their presence in endogenous activated spliceosomes or in a postsplicing snRNP complex.

FIGURE 1.

Protein–protein interactions involving Prp45p, Syf3p, and Prp46p. (A) Prp45p; (B) Syf3p; (C) Prp46p. Numbers represent amino acid positions. Vertical lines delineate minimal regions found in common in overlapping prey fusions.

FIGURE 2.

Effect of Prp45p depletion on pre-mRNA splicing in vivo. (A) Northern analysis of splicing. RNA was extracted from aliquots of the cultures grown for the indicated times under permissive or nonpermissive conditions. RNA (10 μg) was fractionated on a 1% (w/v) formaldehyde gel, blotted to Hybond-N membrane (Amersham), probed with a radiolabeled DNA fragment complementary to exon 1 of the RP28 gene, and analyzed by autoradiography. The positions of the RP28 pre-mRNA and mRNA are indicated. The blot was stripped and reprobed for the intronless PGK gene as a control for loading. (B) Primer extension analysis of splicing of U3 precursor RNA. RNA (10 μg) was used in a primer extension reaction with a radiolabeled oligonucleotide primer complementary to the extreme 5′ end of exon 2 of the U3 snoRNA. As a control, a primer for the intronless U1snRNA was used in the same reaction. The products were resolved on a 6% (w/v) polyacrylamide gel and visualized by autoradiography. The positions of the extension products are indicated.

FIGURE 3.

Coprecipitation of spliceosomes by Prp45p. Whole yeast cell extract (splicing extract) was prepared from cells of strain YMA45/2 grown in galactose-based medium, producing protA:Prp45p. As a control, extract was prepared from the parental wild-type strain carrying vector pNOPPATAIL, producing a double protein A epitope (protA). Splicing (50 μL total volume) was performed using ³²P-labeled actin pre-mRNA. The reactions were stopped and 5 μL were removed as splicing controls (input). The remaining 45-μL samples were mixed with an equal volume of precipitation buffer containing either IgG-agarose beads (I), agarose beads without antibody (B) or protein A-Sepharose beads with prebound anti-Prp8p antibodies (C) and incubated at 4°C for 2 h. Beads were washed in buffer containing 150 mM NaCl, deproteinized, and the RNAs precipitated. The samples from the immunoprecipitations (coIP) as well as from the input samples (5 μL; splicing) were then resuspended in formamide loading buffer, resolved on a 6% (w/v) polyacrylamide gel, and labeled RNAs were visualized by autoradiography. In additional samples, recombinant dominant negative Prp2p was added to the extract prior to splicing (+ Prp2^LATp) and the samples were treated as above. The positions of the RNA species are indicated. (lariat I-E2) Lariat intron-exon2; (LI) Lariat-intron; (E1) exon1.

FIGURE 4.

Coprecipitation of snRNAs by Prp45p. Whole yeast cell extract (splicing extract) was prepared from cells of strain YMA45/2 grown in galactose-based medium, producing protA:Prp45p. As a control, extract was prepared from the parental wild-type strain carrying vector pNOPPATAIL, producing a double protein A epitope (protA). Extracts (50 μL) were mixed with an equal volume of precipitation buffer containing either IgG-agarose beads (I; lanes 3, 6, 9), agarose beads without antibody (B; lanes 4, 7), or protein A-Sepharose beads with prebound anti-Prp8p antibodies (C; lanes 5, 8) and incubated at 4°C for 2 h. The beads were washed in buffer containing 150 mM NaCl (or 75 mM where indicated), deproteinized, and the RNAs precipitated. The samples were resuspended in formamide loading buffer, resolved on a 6% (w/v) polyacrylamide gel, electroblotted to a Hybond-N nylon membrane (Amersham), and probed for U1, U2, U4, U5, and U6snRNAs. Total RNA extracted from 20 μL of extract is shown in lanes 1 and 2.

FIGURE 5.

Effect of Prp46p depletion on cell growth and pre-mRNA splicing. (A) Effect of Prp46p depletion on cell growth. Strains YMA151/2 and BMA64α (WT) were grown at 30°C in minimal medium lacking methionine (inducing conditions for Prp46p production in YMA151/2) or with 7 mM methionine (repressing condition) as indicated. The O.D.₆₀₀ was monitored and cultures were diluted at intervals to maintain logarithmic growth. The indicated O.D.₆₀₀ is therefore the calculated theoretical O.D. (B) Effect of Prp46p depletion on in vivo splicing. RNA (10 μg) extracted from aliquots of the cultures shown in A was used as template in a primer extension reaction using radiolabeled oligonucleotide primers complementary to the extreme 5′ end of exon 2 of the U3 snoRNA and to the (intronless) U1 snRNA as control. The products were resolved on a 6% (w/v) polyacrylamide gel and visualized by autoradiography. The positions of the extension products are indicated. (time [h]) Time point of sample. (Lanes 1,4,7) YMA151/2 minus methionine; (lanes 2,5,8) YMA151/2 plus 7 mM methionine; (lanes 3,6,9) BMA64α plus 7 mM methionine. (Lariat I-E2) lariat intron-exon2.

FIGURE 6.

Prp45p coprecipitates with Prp46p in vitro. (A) ³⁵S-methionine-labeled full-length Prp45p or fragments of the regions indicated were produced in vitro. (B) The in vitro -produced polypeptides were incubated with ATP-depleted yeast splicing extract containing HA₂-tagged Prp46p and with anti-HA antibodies on ice for 1 h before adding protein A-Sepharose beads. The pellets and supernates (1.6%) were analyzed by SDS-PAGE and the ³⁵S-labeled Prp45 polypeptides were visualized by autoradiography.

FIGURE 7.

Prp46p coprecipitates with Prp45p in vitro. (A) ³⁵S-methionine-labeled full-length Prp46p or fragments of the regions indicated were produced in vitro. (B) His₆-Prp45p affinity purified from E. coli extract was incubated with the in vitro produced Prp46 polypeptides on ice for 1 h before adding antipentahistine antibodies bound to protein A-Sepharose beads, or beads alone as a control. The pellets and supernates (1.6%) were analyzed by SDS-PAGE and the ³⁵S-labeled Prp45 polypeptides were visualized by autoradiography.