Splicing Factor Prp8 Governs U4/U6 RNA Unwinding during Activation of the Spliceosome  Andreas N Kuhn, Zairong Li, David A Brow  Molecular Cell  Volume 3, Issue 1, Pages 65-75 (January 1999) DOI: 10.1016/S1097-2765(00)80175-6

Figure 3 U4/U6 Unwinding and U1 Release Are Inhibited by U4-cs1 Splicing complexes were assembled on biotinylated pre-mRNA with wild-type (lanes 1 to 4) or U4-cs1 extracts (lanes 5 to 8) at 16°C for the times indicated. The reactions were stopped with EDTA and then incubated with a streptavidin matrix. After extensive washing, the bound spliceosomal RNAs were eluted under conditions that preserved the U4/U6 base pairing and analyzed as described in the text. In lanes 9 to 11, the U4-cs1 reactions were first incubated at 16°C for 30 min and then shifted to 30°C for the times indicated after addition of excess nonbiotinylated pre-mRNA. Lane 12 shows the result from a control reaction without biotinylated pre-mRNA. The positions of the analyzed spliceosomal RNAs are shown on the right. A longer exposure of the U1 RNA region of the gel is shown at bottom. Molecular Cell 1999 3, 65-75DOI: (10.1016/S1097-2765(00)80175-6)

Figure 6 Model of Structural Rearrangements at the 5 ’ Splice Site during Activation of the Spliceosome (A) RNA-RNA interactions in stalled U4-cs1 spliceosomes. Shown are, from top to bottom, the 5′ end of U1 RNA, the pre-mRNA 5′ splice site, the U6 RNA ACAGA box and stem I region, and the region of U4 RNA containing the U4-cs1 mutation (underlined). Base pairing interactions are depicted by lines (A–U and G–C) or dots (G–U). (B) Model of Prp8 as the regulator of 5′ splice site activation. Prp8 contacts the 5′ splice site region, drawn here in the transitional structure as proposed in (A) with the addition of the interaction between loop 1 of U5 RNA and exon sequences. The DExD/H-box proteins Prp28 and Brr2/Rss1/Slt22/Snu246, proposed to be involved in U1/pre-mRNA and U4/U6 unwinding, respectively (Staley and Guthrie 1998), and controlled by Prp8, are depicted as ovals. Molecular Cell 1999 3, 65-75DOI: (10.1016/S1097-2765(00)80175-6)

Figure 1 U4-cs1 Creates a Reversible Block to Splicing at 16°C In Vitro Splicing extracts from wild-type and U4-cs1 strains were tested for their ability to splice 32P-labeled actin pre-mRNA at 16°C for 30 min (lanes 1 and 2) and at 30°C for 20 min (lanes 3 and 4). Splicing products were analyzed on a denaturing polyacrylamide gel. Lane 5 shows the splicing products of a reaction with U4-cs1 extract that was first incubated at 16°C for 30 min and then allowed to procede for 20 min more at 30°C after addition of unlabeled competitor pre-mRNA. The products of a splicing reaction with U4-cs1 extract at 30°C for 20 min, in which the competitor RNA was added simultaneously with the labeled pre-mRNA, are shown in lane 6. The positions of lariat-intron/exon 2, lariat-intron, pre-mRNA, fully spliced mRNA, and exon 1 (from top to bottom) are indicated schematically on the right. Molecular Cell 1999 3, 65-75DOI: (10.1016/S1097-2765(00)80175-6)

Figure 2 U4-cs1 Blocks Splicing after Formation of the Spliceosome Formation of splicing complexes on 32P-labeled actin pre-mRNA with wild-type or U4-cs1 extract was monitored on a native polyacrylamide gel. Reactions in lanes 1 to 6 were performed at 16°C, stopped at the indicated time points, and loaded on the gel. In lanes 7 to 12, the pre-mRNA was first incubated with U4-cs1 extract at 16°C for 30 min, followed by the addition of excess unlabeled pre-mRNA and either ATP (lanes 7 to 9) or glucose (lanes 10 to 12) and a second incubation at 30°C for the indicated times. The positions of the different splicing complexes are marked on the right. Molecular Cell 1999 3, 65-75DOI: (10.1016/S1097-2765(00)80175-6)

Figure 4 The U4/U6 RNA Unwinding Activity in Splicing Complexes Is Highly Selective for ATP Hydrolysis (A) U4-cs1 splicing complexes were assembled on biotinylated pre-mRNA at 16°C for 30 min and then bound to a streptavidin matrix after depletion of ATP. After one washing step with splicing buffer, the bound complexes were incubated at 30°C for 2, 6, and 20 min with splicing buffer containing 3 mM ATP (lanes 2 to 4) or in the absence of ATP (lanes 5 to 7). Lane 1 shows the assembled complexes before incubation at 30°C. RNA analysis was as in Figure 3. (B) U4-cs1 splicing complexes were assembled and purified as in (A), but then washed either once with splicing buffer (lane 1, low stringency) or three times with the buffer usually used prior to elution (lane 2, high stringency). The washed splicing complexes were incubated with splicing buffer containing 3 mM ATP for 10 min, and the RNAs then analyzed as in (A). (C) Unwinding reactions were performed and analyzed as described in (A) for 10 min with 3 mM of the nucleoside triphosphate indicated. Only the signal for the U4/U6 RNA complex is shown. For quantitation, the intensities were normalized to U2 and then expressed as a percentage of the U4/U6 complex in the reaction without any nucleoside triphosphate. The average of two experiments is shown. Molecular Cell 1999 3, 65-75DOI: (10.1016/S1097-2765(00)80175-6)

Figure 5 Prp8–201 Suppresses U4-cs1 and Exacerbates U4-G14C (A) At the top is a schematic of the Prp8 primary structure. Indicated by shadowing and “+” are the positions of mutations that affect 3′ splice site fidelity and uridine tract recognition, respectively (Umen and Guthrie 1996). The mutation in prp8–201 changes threonine 1861 to proline in a nearly perfectly conserved region of Prp8. Shown is the sequence alignment of amino acids 1842 to 1887 of Saccharomyces cerevisiae (S.c.) Prp8 with the homologous regions from Schizosaccharomyces pombe (S.p., GenBank Z98530), Caenorhabditis elegans (C.e., GenBank L14433), human (H.s., GenBank AB007510), Plasmodium falciparum (P.f., GenBank AL010234), and Trypanosoma brucei (T.b.; Lücke et al. 1997). C.e. and H.s. are identical in this region. Sequence identities are depicted by stars. (B) prp8–201 suppresses the growth defect of U4-cs1. Yeast strain ZRL103 (U4-cs1 as the sole copy of U4 RNA and PRP8 under control of the glucose-repressible GAL1 promoter) was transformed with either YCp50-PRP8 or YCp50-PRP8–201. Cells were plated onto YEPD medium to repress the GAL1 promoter and incubated at 18°C, 30°C, and 37°C as indicated. (C) prp8–201 is synthetically lethal with U4-G14C. Yeast strains with a chromosomal U4 RNA gene disruption, the indicated PRP8 alleles, and a URA3-marked plasmid bearing the wild-type U4 gene were transformed with a HIS3-marked plasmid containing either the wild-type U4 gene (SNR14) or the U4-G14C mutant allele (snr14-G14C) and plated on medium containing 5-FOA to select against the URA3-marked plasmid. Cells were incubated at 30°C for 3 days. Molecular Cell 1999 3, 65-75DOI: (10.1016/S1097-2765(00)80175-6)