Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA

Abstract

Communication between U1 and U2 snRNPs is critical during pre-spliceosome assembly; yet, direct connections have not been observed. To investigate this assembly step, we focused on Prp5, an RNA-dependent ATPase of the DExD/H family. We identified homologs of Saccharomyces cerevisiae Prp5 in humans (hPrp5) and Schizosaccharomyces pombe (SpPrp5), and investigated their interactions and function. Depletion and reconstitution of SpPrp5 from extracts demonstrate that ATP binding and hydrolysis by Prp5 are required for pre-spliceosome complex A formation. hPrp5 and SpPrp5 are each physically associated with both U1 and U2 snRNPs; Prp5 contains distinct U1- and U2-interacting domains that are required for pre-spliceosome assembly; and, we observe a Prp5-associated U1/U2 complex in S. pombe. Together, these data are consistent with Prp5 being a bridge between U1 and U2 snRNPs at the time of pre-spliceosome formation.

Figure 1

SpPrp5 is required for the formation of S. pombe complex A. (A) Comparison of human (upper) and fission yeast (lower) Prp5 to S. cerevisiae Prp5. Eight conserved motifs of DExD/H family ATPases and the Q motif of the DEAD subfamily are indicated by black boxes (Caruthers and McKay, 2002; Tanner et al, 2003). Other regions of similarity are boxed in gray. ^*, DPLD motif unique to Prp5 homologs; RS/RD, region rich in RS, RD, and RE dipeptides. (B) (Upper) Schematic of SpPrp5-TAP fusion protein. CBP, calmodulin-binding peptide; TEV, TEV protease cleavage site; prot. A, two copies of the protein A IgG binding domain. (Lower) Analysis of SpPrp5 depletion. Untagged extracts (WT; lane 1), mock-depleted WT (lane 2), SpPrp5-TAP tagged (lane 3), and SpPrp5-TAP depleted (lane 4) were separated on 10% SDS–PAGE, Western blotted, and probed for TAP and for U2-SF3b155 as a control. Parallel Northern analysis indicated that no snRNAs were codepleted under these conditions (not shown). (C) GST-SpPrp5 reconstitutes SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with ³²P-labeled pre-mRNA –/+ATP at 30°C as indicated. GST alone, GST-SpPrp5, or Prp43p were added to depleted extracts. Samples were separated on a native 4% polyacrylamide gel, and visualized by phosphorimaging. A, U2 snRNP complexes containing pre-mRNA; H, nonspecific complexes. (D) SpPrp5-TAP is present in complexes with pre-mRNA. Extracts prepared from strains containing SpPrp5-TAP, U2-A′-TAP, or no tagged protein were incubated with ³²P-labeled pre-mRNA –/+ATP as indicated, and complexes were affinity selected using IgG-sepharose to bind the TAP moiety. Copurifying pre-mRNA (upper); IgG-selected TAP-tagged proteins detected by Western analysis for the TAP tag (lower).

Figure 2

ATP hydrolysis by SpPrp5 is required for the formation of S. pombe complex A. (A) Mutations introduced into GST-Prp5 proteins: in motif I, GKT was changed to GAT (K468A) or GKA (T469A); in motif II, DEAD was changed to AEAD (D575A) or DAAD (E576A); and in motif III, SAT was changed to SAA (T608A). (B) UV crosslinking of GST-Prp5 and mutant proteins to [γ-³²P]ATP. (C) Comparison of ATPase activities of GST-SpPrp5 and mutated proteins. Values are the average from at least two parallel reactions. GST-SpPrp43 (not shown) had a similar level of ATPase activity as ScPrp43-His. (D) Ability of ATPase mutants to complement complex A formation in SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with ³²P-labeled pre-mRNA –/+ATP; either GST-SpPrp5 or mutant proteins were added to depleted extracts, as indicated. WT^*, control protein whose coding sequence was altered to contain a new restriction site, but without change in amino-acid sequence. (E) ATPase mutants bind U1 and U2 snRNPs and compete effectively with WT SpPrp5 protein. GST-Prp5 proteins (WT, GAT, DAAD, SAA, or GST alone) were added to SpPrp5-TAP extract and incubated for 30 min; then snRNPs were affinity selected using the TAP tag. GKA and AEAD mutants also effectively competed for U1 and U2 binding (not shown).

Figure 3

Human and S. pombe Prp5 associate with U1 and U2 snRNPs. (A) SpPrp5-TAP association with pre-mRNA depends on both U1 and U2 snRNPs. Extracts prepared from strains containing SpPrp5-TAP, U1-A-TAP, or U2-A′-TAP were treated with RNase H alone (lane 2) or with RNase H and oligonucleotides targeting nt 1–14 of U1 (lane 3), nt 28–42 of U2 (lane 4), or nt 21–69 U6 snRNA as a control (lane 5). Extracts were then incubated with ³²P-labeled pre-mRNA +ATP, and complexes were affinity selected using IgG-sepharose to bind the TAP moiety. Copurifying pre-mRNA was analyzed by 10% PAGE (upper). Northern analysis of input snRNAs from SpPrp5-TAP extract after RNase H degradation (lower). The U1-A-TAP and U2-A′-TAP extracts were analyzed in parallel and showed similar levels of snRNA degradation (data not shown). (B) Affinity selection of snRNA associated with SpPrp5. Untagged (WT) or SpPrp5-TAP extracts were incubated at 30°C for 30 min and then with IgG-sepharose beads. Lanes 1 and 4, 1/4 of input. Copurifying snRNAs were analyzed by Northern blotting. (C) Affinity selection of U1 and U2 snRNP proteins by SpPrp5. Extracts from doubly tagged S. pombe strains containing SpPrp5-TAP and either U1-70K-3HA or U2-A′-3HA were incubated with IgG-sepharose. Copurifying proteins were analyzed by Western blotting using anti-HA antibodies. Lanes 1–2 and 7–8: 1/4 of the input extracts. Lanes 3–4 and 9–10: IgG-selected material from cells lacking any TAP-tagged protein as a control. Lanes 5–6 and 11–12: HA-tagged proteins copurifying with SpPrp5-TAP. (D) hPrp5 co-IPs U1 and U2 snRNPs. HeLa nuclear extract was incubated with –/+ATP as indicated, and then with protein A-bound antibodies. 1/20 of input (lane 1), beads alone (lanes 2–3), anti-hPrp5 #1 against a C-terminal peptide (lanes 4–5), anti-hPrp5 #2 against the internal DPLD motif (lanes 6–7).

Figure 4

SpPrp5 interacts independently with U1 snRNP and with U2 snRNP. (A) Four schemes for possible interaction of Prp5 with U1 and U2 snRNPs. See text for details. Data in panel B argue against schemes I or II; and data of panels C and D and in Figure 5 support scheme III. Note that schemes III and IV are not mutually exclusive. (B) Northern analysis of SpPrp5-TAP affinity-purified snRNAs after RNase H-targeted degradation. Either untagged (WT) or SpPrp5-TAP extracts were treated with RNase H alone (lanes 1 and 3) or with RNase H and oligonucleotides targeting U6 snRNA as a control (lanes 2 and 4), or regions of U1 or U2 as indicated (lanes 5–8). SpPrp5-TAP-associating snRNAs were then affinity selected and analyzed by Northern blots sequentially probed for U1, U2, and U6. Truncated U1 and U2 indicate RNase H cleavage products. Aliquots of each sample were also analyzed for the amount of affinity-selected SpPrp5-TAP (Western, lower panel). (C) Analysis of distribution of Prp5 in S. pombe. Affinity selection of snRNAs associated with fractionated SpPrp5. SpPrp5-TAP extract was fractionated through a 10–30% glycerol gradient. Fractions were mixed with IgG-sepharose beads. Copurifying snRNAs were separated on a urea-denaturing gel and detected by Northern analysis (lower). Fractions were also analyzed by Western blots probed for SpPrp5-TAP (upper). Positions of 18S and 25S ribosomal RNAs from parallel gradients are indicated. (D) Affinity selection of a U2 snRNP protein supports the presence of U1/U2 di-snRNP. Extracts from either WT or a strain containing U2-A′-TAP were incubated with –/+ATP as indicated and then with IgG-sepharose. Copurifying snRNAs were analyzed as in Figure 3B.

Figure 5

Different domains of SpPrp5 interact with U1 and U2 snRNP. (A) Schematic of GST-Prp5 deletion proteins. (B) ATPase activities of GST-SpPrp5 and deletion constructs, expressed and purified from E. coli. Protein A: full-length SpPrp5; B: aa171–1014; C: aa272–1014; D: aa427–1014; E: aa1–170; F: aa171–426. (C) Affinity selection of snRNA by GST-SpPrp5. Extracts were incubated with full-length or deleted SpPrp5 proteins as indicated, and then with glutathione-sepharose beads. Copurifying snRNAs were analyzed by Northern blotting. (D) Ability of deletion mutants to complement complex A formation in SpPrp5-TAP depleted extracts. Intact and depleted SpPrp5-TAP extracts were incubated with ³²P-labeled pre-mRNA as indicated. Either GST-FL-SpPrp5 (A) or deletion proteins (B–F) were added to depleted extracts.

Figure 6

Schematic models of Prp5 bridging interaction with snRNPs during pre-spliceosome assembly. (Left) U1 and U2 bind substrate and are subsequently bridged by Prp5. (Right) Prp5 bridges U1 and U2 snRNPs prior to substrate binding. Hydrolysis of ATP by Prp5 is required for stable binding of U2 snRNP around the branch region. See text for details.