An Allosteric Network for Spliceosome Activation Revealed by High-Throughput Suppressor Analysis in Saccharomyces cerevisiae

Abstract

Selection of suppressor mutations that correct growth defects caused by substitutions in an RNA or protein can reveal functionally important molecular structures and interactions in living cells. This approach is particularly useful for the study of complex biological pathways involving many macromolecules, such as premessenger RNA (pre-mRNA) splicing. When a sufficiently large number of suppressor mutations is obtained and structural information is available, it is possible to generate detailed models of molecular function. However, the laborious and expensive task of identifying suppressor mutations in whole-genome selections limits the utility of this approach. Here I show that a custom targeted sequencing panel can greatly accelerate the identification of suppressor mutations in the Saccharomyces cerevisiae genome. Using a panel that targets 112 genes encoding pre-mRNA splicing factors, I identified 27 unique mutations in six protein-coding genes that each overcome the cold-sensitive block to spliceosome activation caused by a substitution in U4 small nuclear RNA. When mapped to existing structures of spliceosomal complexes, the identified suppressors implicate specific molecular contacts between the proteins Brr2, Prp6, Prp8, Prp31, Sad1, and Snu114 as functionally important in an early step of catalytic activation of the spliceosome. This approach shows great promise for elucidating the allosteric cascade of molecular interactions that direct accurate and efficient pre-mRNA splicing and should be broadly useful for understanding the dynamics of other complex biological assemblies or pathways.

Keywords: U4 snRNA; pre-mRNA splicing; spliceosome activation; suppressor selection; targeted sequencing.

Figure 1

U4-cs1 may compete with the intron 5′ splice site for binding to the U6 ACAGA box (underlined). In the inset, the dotted box indicates the region of the U4/U6 duplex that is enlarged. The U4-cs1 substitution is shown in red; the wild-type sequence is AAA. The yeast consensus intron 5′ splice site is shown as it pairs with U6 in the catalytically competent spliceosome. Potential competing base pairs created or stabilized by the U4-cs1 mutation are shown in red.

Figure 2

Distinct U4-cs1-suppressor mutations arise in a single culture. A plate from the selection for cold-resistant U4-cs1 colonies is shown. Colonies that were picked are labeled with their strain designation (DAB1XX) and mutation, if any, in the 112 genes sequenced.

Figure 3

Location of suppressor substitutions in Prp8 in the yeast B complex. The cryo-EM structure is from Plaschka et al. (2017) (PDB: 5nrl). The major domains of Prp8 are indicated as follows: NTD1 and NTD2, RT, Linker, Endonuclease-like (Endonuc.), RNase H-like (RNase H), and Jab1/MPN (Galej et al. 2013; Nguyen et al. 2016; Bertram et al. 2017). Residues altered by U4-cs1-suppressors identified in Kuhn and Brow (2000) or this study (yellow, Table S6) are labeled if clearly visible. Sites of identified prp28-1 suppressors (Price et al. 2014) and brr2-1 suppressors (Kuhn et al. 2002) are colored green and red, respectively. See Table S6 for substitutions. This and subsequent figures were created using the PyMOL Molecular Graphics System, version 1.8.2.1 (Schrödinger, LLC).

Figure 4

U4-cs1-suppressors in Brr2 cluster together in its N-terminal PWI domain. The U4-cs1-suppressor sites in Brr2’s PWI domain (yellow spheres) face, but do not contact, its catalytic N-terminal helicase domain and regulatory C-terminal helicase domain. Linkers that join the PWI domain to the rest of Brr2 are disordered; dotted lines indicate the connectivity. The structure is from Absmeier et al. (2017) (PDB: 5m52).

Figure 5

A subset of U4-cs1-suppressor mutations map to the Sad1-Brr2-Snu114-Prp8 interface in a model of the human U4/U6.U5 tri-snRNP. Amino acid residues changed in certain U4-cs1-suppressor strains are shown in spheres of the same color as the parent protein, except for Brr2 where the residues are yellow. Some are labeled with the residue number and wild-type identity. Four interfaces that harbor suppressor substitutions are marked by ellipses and comprise portions of the following protein domains: (1) Prp8(HB/RT1)-Sad1(CTD)-Snu114(NTD), (2) Prp8(RT2)-Sad1(mid)-Snu114(D3), (3) Sad1(ZnF-UBP)-Snu114(D2/3/4a), and (4) Sad1(ZnF-UBP)-Brr2(PWI). The model is based on a 7-Å cryo-EM structure of the human tri-snRNP (Agafonov et al. 2016) and was provided by Holger Stark and Reinhard Lührmann. Equivalent residues in yeast and human were assigned based on sequence alignment. Portions of the proteins that do not directly participate in the Sad1-Brr2-Snu114-Prp8 interface are not shown.

Figure 6

Retention of Sad1 in the B complex is expected to prevent engagement of U4 by the Brr2 N-terminal helicase domain. Shown are cryo-EM structures of selected components of the human tri-snRNP (left, as in Figure 5) and the yeast B complex (right; Plaschka et al. 2017; PDB: 5nrl). Residues in which U4-cs1-suppressor substitutions were obtained (Table 1 and Table S6) are indicated by yellow spheres. The C-terminal Jab1/MPN domain of Prp8 is omitted from the human tri-snRNP structure. In the human tri-snRNP, the catalytic N-terminal helicase domain (NHD) of Brr2 is far from U4/U6 Stem I (green and red), presumably due to stable contacts between its PWI domain and Sad1. In the yeast B complex, Sad1 is absent and Brr2 has rotated ∼180°, along with the Prp8 RNase H-like domain, and has engaged the single-stranded region of U4 downstream of U4/U6 Stem I in the Brr2-NHD active site. The Brr2 PWI domain is not modeled in the yeast B complex. The intron 5′ splice site is shown paired with the U6 ACAGA stem-loop (Plaschka et al. 2017), upstream of its final location at the U6 ACAGA box. CHD, C-terminal helicase domain; Endo, endonuclease-like.

Figure 7

A subset of U4-cs1-suppressors in Prp8 colocalize with Prp28 in the human tri-snRNP. The two RecA domains of human (h) Prp28 (gray) contact residues in Prp8 that, when mutated in yeast, suppress the cold sensitivity of prp28-1 (green) or U4-cs1 (yellow) strains. Parts of the hPrp8 NTD1 and linker/endonuclease-like (linker/endo) domains are shown, and residues are indicated with yeast (y) numbering. The N-terminal 351 residues of hPrp28 are not modeled, but the C-terminal end of the Prp28-NTD (labeled) is adjacent to prp28-1-suppressor substitutions in Prp8. Model coordinates were kindly provided by Holger Stark and Reinhard Lührmann.

Figure 8

A subset of U4-cs1-suppressors colocalize with brr2-1 suppressors in the RT domain of Prp8, at an interface with Prp6, and Prp31. (Top) The view is similar to that of the top of the yeast B complex structure shown in Figure 6, with the removal of U4/U6 and addition of Prp6 and Snu66. Selected suppressors of U4-cs1 (yellow) and brr2-1 (red) are labeled with their wild-type and mutant identities. Prp8-V1098D suppress both mutations (Kuhn et al. 2002). U4-cs1-suppressors in the β-hairpin of the Prp8 RNase H-like domain are in residues 1860–1862, 1872, and 1875, and appear to contact Snu66. (Bottom) Same as figure above, but with Prp31 added. The sole U4-cs1-suppressor in Prp31 is labeled. Note that Prp31 appears to contact several of the U4-cs1 and brr2-1 suppressors.

Figure 9

Several U4-cs1-suppressor substitutions in Prp8 map to a potential IP6-binding site. The model shown is of the yeast C* spliceosome (Fica et al. 2017; PDB: 5mq0). Prp8 is lavender with U4-cs1-suppressor sites in yellow. The inositol group is green and the attached phosphates are orange and red. Modeled distances between atoms indicated by yellow dotted lines are in Ångstroms and are consistent with hydrogen bonds.