Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5′-UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5′ splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein–RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.     

Structure-function analysis and genetic interactions of the yeast branchpoint binding protein Msl5

Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.     

Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint

Splicing of pre-mRNA is initiated by binding of U1 to the 5′ splice site and of Msl5-Mud2 heterodimer to the branch site (BS). Subsequent binding of U2 displaces Msl5-Mud2 from the BS to form the prespliceosome, a step governing branchpoint selection and hence 3′ splice site choice, and linking splicing to myelodysplasia and many cancers in human. Two DEAD-box proteins, Prp5 and Sub2, are required for this step, but neither is stably associated with the pre-mRNA during the reaction. Using BS-mutated ACT1 pre-mRNA, we previously identified a splicing intermediate complex, FIC, which contains U2 and Prp5, but cannot bind the tri-snRNP. We show here that Msl5 remains associated with the upstream cryptic branch site (CBS) in the FIC, with U2 binding a few bases downstream of the BS. U2 mutants that restore U2-BS base pairing enable dissociation of Prp5 and allows splicing to proceed. The CBS is required for splicing rescue by compensatory U2 mutants, and for formation of FIC, demonstrating a role for Msl5 in directing U2 to the BS, and of U2-BS base pairing for release of Prp5 and Msl5-Mud2 to form the prespliceosome. Our results provide insights into how the prespliceosome may form in normal splicing reaction.     

Snf1p-dependent Spt-Ada-Gcn5-acetyltransferase (SAGA) recruitment and chromatin remodeling activities on the HXT2 and HXT4 promoters

We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p. In addition, we found that the Gcn5p subunit of the SAGA complex preferentially acetylates histone H3K18 on the HXT2 and HXT4 promoters and that Gcn5p activity is required for removal of histone H3 from the HXT4 promoter TATA region. In contrast, histone H3 removal from the HXT2 promoter does not require Gcn5p. In conclusion, although similar protein complexes are involved, induction of HXT2 and HXT4 displays important mechanistic differences.     

Chromatin remodelling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding

The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively. Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo. These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site. However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions. Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding. These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.     

Global role for chromatin remodeling enzymes in mitotic gene expression

Regulation of eukaryotic gene expression requires ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, and histone acetyltransferases, such as Gcn5p. Here we show that SWI/SNF remodeling controls recruitment of Gcn5p HAT activity to many genes in late mitosis and that these chromatin remodeling enzymes play a role in regulating mitotic exit. In contrast, interphase expression of GAL1, HIS3, PHO5, and PHO8 is accompanied by SWI/SNF-independent recruitment of Gcn5p HAT activity. Surprisingly, prearresting cells in late mitosis imposes a requirement for SWI/SNF in recruiting Gcn5p HAT activity to the GAL1 promoter, and GAL1 expression also becomes dependent on both chromatin remodeling enzymes. We propose that SWI/SNF and Gcn5p are globally required for mitotic gene expression due to the condensed state of mitotic chromatin.     

Interactions of the yeast SF3b splicing factor

The U2 snRNP promotes prespliceosome assembly through interactions that minimally involve the branchpoint binding protein, Mud2p, and the pre-mRNA. We previously showed that seven proteins copurify with the yeast (Saccharomyces cerevisiae) SF3b U2 subcomplex that associates with the pre-mRNA branchpoint region: Rse1p, Hsh155p, Hsh49p, Cus1p, and Rds3p and unidentified subunits p10 and p17. Here proteomic and genetic studies identify Rcp10p as p10 and show that it contributes to SF3b stability and is necessary for normal cellular Cus1p accumulation and for U2 snRNP recruitment in splicing. Remarkably, only the final 53 amino acids of Rcp10p are essential. p17 is shown to be composed of two accessory splicing factors, Bud31p and Ist3p, the latter of which independently associates with the RES complex implicated in the nuclear pre-mRNA retention. A directed two-hybrid screen reveals a network of prospective interactions that includes previously unreported intra-SF3b contacts and SF3b interactions with the RES subunit Bud13p, the Prp5p DExD/H-box protein, Mud2p, and the late-acting nineteen complex. These data establish the concordance of yeast and mammalian SF3b complexes, implicate accessory splicing factors in U2 snRNP function, and support SF3b contribution from early pre-mRNP recognition to late steps in splicing.