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

Abstract

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.

Figure 1.

Domain organization and primary structure of yeast Msl5. The 476-amino acid Msl5 polypeptide is depicted in the top panel as a linear array with the N-terminus at left and the C-terminus at right and the known or imputed domains drawn as cylinders spanning their segments of the primary structure. The amino acid sequence of S. cerevisiae Msl5 (Sce; Genbank accession NP_013217) from amino acids 11–476 is aligned to that of the homologous 566-amino acid polypeptide from Aspergillus fumigatus (Afu; Genbank accession XP_754535) in the bottom panel. Positions of amino acid site chain identity/similarity are denoted by dots above the sequence. The segments corresponding to the various domains are underlined according to the color scheme in the top panel. Gaps in the alignment are denoted by dashes. Forward and reverse arrowheads indicate the boundaries of the N-terminal and C-terminal truncations, respectively. Vertical lines and brackets above the sequence signify the amino acids subjected to single-alanine or alanine-cluster mutagenesis.

Figure 2.

Structure-guided mutations of the Mud2-Msl5 interface. (A) Stereo view of the crystal structure of the RRM3 domain of human U2AF65 (colored green) bound to a peptide ligand derived from human SF1 (colored gray) as reported in pdb 1OPI (30). The human RRM3 domain is homologous to the RRM3 domain of yeast Mud2. Amino acids in the RRM3 domain that comprise the SF1 binding site are shown as stick models and numbered according to their equivalent positions in Mud2. The tryptophan side chain (Trp22) of human SF1 peptide that is critical for binding to human U2AF65 is shown as a gray stick model. The putative equivalent position in yeast Msl5 is Trp35. (B) Alignment of the primary structures of the RRM3 domains of S. cerevisiae Mud2 (Accession AAA64215), S. pombe U2AF59 (Accession NP_595396) and Homo sapiens U2AF65 (Accession P26368). Positions of side chain identity/similarity in all three proteins are indicated by dots above the alignment. The secondary structure elements are depicted below the alignment (arrows for β strands and bars for α helices). The RNP2 and RNP1 motifs are highlighted in gray boxes. The five RNP2 and RNP1 residues of Mud2 that were mutated previously to alanine (16) are indicated by vertical lines above the alignment. [The equivalents in human U2AF65 are colored magenta in (A)]. The putative branchpoint binding protein-interacting residues of Mud2—Glu443, Glu445, Leu498 and Phe503—that were subjected to mutation, pair-wise and en masse, are denoted by bracketed arrows.

Figure 3.

Structure-guided mutations of the KH and QUA2 domains of yeast Msl5. The top panel shows a stereo view of the NMR structure (; pdb 1K1G) of the KH and QUA2 domains of human SF1, colored green and magenta, respectively, bound to an RNA oligonucleotide 5′-AUACUAACAA that contains the consensus yeast intron branchpoint (underlined). The RNA is rendered as a stick model with gray carbons, except for the branchpoint adenosine (yellow carbons) that transesterifies to the 5′ splice site to form the lariat intermediate. SF1 side chains that comprise the RNA docking site and are conserved in yeast Msl5 are depicted as stick models with beige carbons and are numbered according to their positions in yeast Msl5. In the bottom panel, the amino acids sequences of the KH-QUA2 segments of S. cerevisiae (Sce) Msl5 and Homo sapiens (Hsa) SF1 (Genbank accession NP_004621) are aligned. The secondary structure elements are depicted below the alignment (arrows for β strands and cylinders for α helices). The conserved residues chosen for mutagenesis are highlighted in yellow boxes. The green box highlights a dipeptide in the QUA2 domain (KR in yeast; RK in human) that was suggested (26) to be a determinant of yeast versus human branchpoint sequence recognition; this dipeptide in Msl5 was subjected to mutagenesis presently.

Figure 4.

Network of genetic interactions of yeast Msl5 domains.