www.sciencemag.org/cgi/content/full/science.aag2235/DC1

Supplementary Materials for

Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution

Ruixue Wan, Chuangye Yan, Rui Bai, Gaoxingyu Huang, Yigong Shi*

*Corresponding author. Email: shi-lab@tsinghua.edu.cn

Published 21 July 2016 on Science First Release

DOI: 10.1126/science.aag2235

This PDF file includes:

Materials and Methods

Figs. S1 to S14

Tables S1 to S4

Full Reference List

Wan & Yan et al

Materials and Methods

Purification of the spliceosomal complexes

Purification of the spliceosomal complexes from the yeast Saccharomyces cerevisiae

(S. cerevisiae) was as described (42).

EM data acquisition and processing

Preparation of the cryo-electron microscopy (cryo-EM) sample and acquisition of the

EM micrographs were as described (42). After the manual check procedure, a data set

of 761,767 particles was produced for further processing (42).

Image processing

The overall shape and appearance of the previously characterized spliceosomal

complexes (26, 30) served as a useful guide for our visual judgment of which 3D class

averages may correspond to the Bact, C, and ILS complexes. Our preliminary analysis

suggested that the spliceosomal C and ILS complexes may be represented by about

34.5 percent of the total particles (42). The same strategy of 3D classification as that

used for the reconstruction of the Bact spliceosome (42) was applied to the

spliceosomal C complex. To avoid the problem of discarding good particles, we

simultaneously performed three independent 3D classifications (K=5, 6, and 7) (Fig.

S1). Then we merged all seven classes that appear to represent the C complex; using

an in-house script, we removed the duplicated particles according to the unique index

2

Wan & Yan et al

of each particle given by RELION (69). This procedure resulted in the selection of

442,876 particles, representing 58.1 percent of the total. The same procedure was

repeated one more time, yielding 161,066 good particles (21.1 percent of the total).

Following auto-refinement, these 161,066 particles gave a reconstruction with an

average resolution of 3.95 Å. After per-particle motion correction and radiation-

damage weighting (known as particle polishing) (70), these polished particles give a

reconstruction with an improved average resolution of 3.41 Å following auto-

refinement (Figs. S1 & S2). The core regions of the C complex display excellent EM

density map; but some of the peripheral regions only have continuous density after the

maps were low-pass filtered to 10 Å (Fig. S2D).

To improve the density at the peripheral regions, we performed a third round

3D classification (K=5) without alignment using the refined polished particles. 80,367

particles in one major class, representing 49.9 percent of the total input, gave an

average resolution of 3.65 Å (Figs. S3 & S4). Although the resolution is a bit lower,

the quality of the EM density maps in some regions is better than that of the 3.41 Å

reconstruction. Finally, an additional round of 3D classification was performed, and

one class with 20,686 particles was identified and gave an average resolution of 4.6

Å. This map gave a better density for the U2 snRNP region (Fig. S3). The angular

distribution of the particles used for the reconstruction of the C complex at 3.65 Å

resolution is reasonable (Fig. S4A), and the refinement of the atomic coordinates did

not suffer from severe overfitting (Fig. S4B). The resulting density maps show clear

3

Wan & Yan et al

features for the secondary structural elements and amino acid side chains for most

protein components of the C complex (Figs. S5-S7). The RNA elements and their

interacting proteins are also well defined by the EM density maps (Fig. S8-S11).

Reported resolutions are calculated on the basis of the gold-standard FSC

0.143 criterion (Fig. S4C), and the FSC curves were corrected for the effects of a soft

mask on the FSC curve using high-resolution noise substitution (71). Prior to

visualization, all density maps were corrected for the modulation transfer function

(MTF) of the detector, and then sharpened by applying a negative B-factor that was

estimated using automated procedures (72). Local resolution variations were

estimated using ResMap (73).

Model Building and refinement

Due to a wide range of resolution limits for the various regions of the spliceosomal C

complex, we combined de novo model building and homologous structure modeling

to generate an atomic model (Tables S1-S4). Identification and docking of the

components of the C complex were facilitated by the structures of the ILS complex at

3.6 Å resolution (10, 40) and the Bact complex at 3.5 Å resolution (42). The protein

components derived from the associated PDB accession code (3JB9 for the ILS

complex from S. pombe) are summarized in Table S2. These structures were docked

into the density map using COOT (74) and fitted into density using CHIMERA (67).

4

Wan & Yan et al

The structure of the spliceosomal Bact complex (42) greatly facilitates the

atomic modeling of 13 protein components. The atomic coordinates of Prp8, Snu114,

Clf1, Cef1, Bud31, Cwc2, Cwc15, Ecm2, Prp45, Prp46, Cwc21, Cwc22, and Prp17

from the cryo-EM structure of the Bact complex (42) were individually docked into the

density maps and manually rebuilt using COOT (74).

For each of the remaining four protein components that lacks a homologous

structure, de novo model building was performed, annotated with the label “De novo

building” in Table S2. These protein components include the NTC proteins Syf2 and

Isy1, and the splicing factors Yju2 and Cwc25. The chemical properties of proteins

and amino acids were considered to facilitate model building. Sequence assignment

was guided mainly by bulky residues such as Phe, Tyr, Trp and Arg. Unique patterns

of sequences were exploited for validation of residue assignment.

The RNA sequence assignment was greatly aided by the structure of the

spliceosomal Bact complex (42). The RNA sequences were manually built using

COOT (74). The RNA nucleotides, together with all protein components, were refined

using REFMAC in reciprocal space (75). To further improve the geometries of the

RNA nucleotides, the RNA elements alone were adjusted using RCrane (76). The

conformations of the RNA components were further refined using phenix.erraser (77).

ERRASER is a Rosetta program for modeling RNA nucleotides into density.

5

Wan & Yan et al

On the basis of the EM density maps, we identified five metal ions that are

bound by nucleotides in the ISL of U6 snRNA. On the basis of previous biochemical

characterizations (1, 5, 8, 9), these metal ions were tentatively assigned as Mg2+. In all

cases, the local maxima of the EM density that may correspond to ions are 2.0–2.4 Å

away from the oxygen atoms of the phosphate groups, consistent with the metrics for

Mg2+ coordination (78-82). In contrast, K+ is usually measured at 2.8–3.5 Å from the

coordinating ligands (78, 79, 83). Therefore, the densities seen here are likely those of

Mg2+. Despite the high likelihood, we acknowledge that at the reported resolution we

cannot unambiguously assign these metal ions to Mg2+. In addition, we cannot

conclusively differentiate Mg2+ from water molecules, although water molecules

should be much less visible in the EM density maps.

Structure refinement of the individual protein was carried out using

phenix.real_space_refine application in PHENIX in real space (84) with secondary

structure and geometry restraints to prevent over-fitting. The final overall model was

refined against the overall 3.65 Å map using REFMAC in reciprocal space (75), using

secondary structure restraints that were generated by ProSMART (85). Overfitting of

the overall model was monitored by refining the model in one of the two independent

maps from the gold-standard refinement approach, and testing the refined model

against the other map (86) (Fig. S4B).

Protein structures in the C complex were individually validated through

6

Wan & Yan et al

examination of their Molprobity scores, statistics of Ramachandran plots, and

EMRinger scores (Table S3). Only protein structures that were solved by homology

modeling or de novo building in Table S2 are included for this practice. For obvious

reasons, those structures that were fitted into the cryo-EM density maps by rigid-body

docking were omitted for such model validation. Molprobity scores were calculated as

described (87) . EMRinger scores were calculated as described (88). EMRinger is a

side chain–directed model and map validation tool for cryo-EM structure

determination. EMRinger evaluates how precise an atomic model is fitted into the

cryo-EM map during refinement. EMRinger scores should be above 1.0 for well-

refined structures with maps in the 3- to 4-Å range. The RNA nucleotides in the C

complex were validated directly by the Molprobity server, and the results are shown

in Table S4.

7

Wan & Yan et al

Table S1. Cryo-EM data collection and refinement statistics. Data collection EM equipment FEI Titan Krios Voltage (kV) 300 Detector Gatan K2 Pixel size (Å) 1.306 Electron dose (e-/Å2) 45.6 Defocus range (µm) 1.6~2.6 Reconstruction Software RELION 1.4 Number of used Particles 161,066 / 80,367 Accuracy of rotation (˚) 0.441 / 0.494 Accuracy of translation (pixels) 0.402 / 0.405 Final Resolution (Å) 3.41 / 3.65 Model building software Coot & Rosetta Refinement Software Phenix & Refmac Map sharpening B-factor (Å2) -83.4 Average Fourier shell correlation 0.893 R-factor 0.245

Model composition Protein residues 8,587 RNA nucleotides 434 GTP 1 Validation R.m.s deviations Bonds length (Å) 0.010 Bonds Angle (˚) 1.343 Ramachandran plot statistics (％) Preferred 92.48 Allowed 5.94 Outlier 1.59

8

Wan & Yan et al

Table S2 Summary of model building for the yeast spliceosomal C complex. Molecule Length Domain/Region PDB code Modeling Resolution (Å)

U5 snRNP

U5 snRNA 214 28:183 From Bact Homology modeling 2.9~4.0

Prp8

2413

N-terminal Domain (127:839) RT finger/palm (840:1253)

Thumb/X (1254:1377) Linker (1378:1650)

Endonuclease (1651:1829) RNaseH-like (1830:2085) Jab1/MPN (2148-2398)

From Bact

Homology modeling

Not modeled

2.9~4.0

~4.0

Snu114 984 67:975 From Bact Homology modeling 2.9~4.0 SmB1 SmD1 SmD2 SmD3 SmE1 SmF1 SmG1

196 146 110 101 94 86 77

Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold

From Bact

Rigid docking

4.0~6.0

U6 snRNP U6 snRNA 112 nt 1:106 From Bact Homology modeling 2.9~4.0 U2 snRNP

U2 RNA 1175 nt 1:48 53:107

109:191

From Bact

- 3JB9

Homology modeling RNA duplex

Rigid Docking

2.9~4.0 5.0~8.0 5.0~8.0

Lea1 238 LRR domain 3JB9 Rigid Docking 5.0~8.0 Msl1 111 RRM domain 3JB9 Rigid Docking 5.0~8.0 SmB1 SmD1 SmD2 SmD3 SmE1 SmF1 SmG1

196 146 110 101 94 86 77

Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold

3JB9

Rigid docking

5.0~8.0

NTC/Prp19 Complex

Clf1 687 TPR domain (40:275) -

From Bact 3JB9

Homology modeling Rigid docking

3.0~4.5 ~30

Syf1 859 - 3JB9 Rigid docking ~30

Cef1 590 Myb Domain (9:111) 145:253

-

From Bact 3JB9 3JB9

Homology modeling Homology modeling

Rigid docking

3.0~3.5 3.0~3.5

~30 Prp19 503 - 3JB9 Rigid docking ~30 Syf2 215 92:211 - De novo building 3.5~4.0

Isy1 235 2:96 - De novo building 3.5~4.0 Snt309 175 - 3JB9 Rigid docking ~30

NTC-Related proteins

Bud31 157 1:157 From Bact Homology modeling 3.0~3.5

Cwc2 339 1:261 Homology modeling 3.0~4.0

Cwc15 175 3:41/127:175 Homology modeling 3.0~4.0 Ecm2 364 RRM domain (3:288) Homology modeling 3.0~5.0

Prp45 379 34:247 Homology modeling 3.0~4.0 Prp46 451 WD40 domain (111:447) Homology modeling 2.9~3.5

Known Splicing Factors

Cwc21 135 2:28 From Bact Homology modeling 3.0~4.0 Cwc22 577 MIF4G domain (11:263)

MA3 domain (279:485) From Bact Rigid docking

Homology modeling 4~10

3.0~3.5 Cwc25 179 N-terminal domain (2:42) - De novo building 2.8~3.2

Yju2 278 2:116 - De novo building 3.0~4.0 Step2 proteins

Prp17 455 50:75 3JB9 Homology modeling 3.0~4.2

Pre-mRNA Pre-mRNA - 57nt From Bact De novo building 2.9~4.5

9

Wan & Yan et al

Table S3 Summary of model validation for individual proteins of the yeast C complex (Proteins solved by homology modeling or de novo building in Table S2 are included here). *EMRinger: side chain–directed model and map validation tool for 3D cryo-electron microscopy

that can assesses the precise fitting of an atomic model into the map during refinement. To validate the model-to-map correctness of atomic models from cryo-EM, refinement should result in EMRinger scores above 1.0 for well-refined structures with maps in the 3- to 4-Å range.

Molecule Molprobity Scores

Ramachandran plot statistics (％) EMRinger* Score

Preferred Allowed Outlier Prp8 2.16 92.44 6.04 1.52 3.13

Snu114 2.19 91.86 6.88 1.26 2.97 Clf1 2.24 93.28 6.72 0.00 2.57 Syf2 1.71 96.94 2.04 1.02 1.70 Isy1 2.35 86.02 12.90 1.08 1.65 Cef1 2.10 93.27 4.33 2.40 2.54

Bud31 2.05 90.32 8.39 1.29 1.72 Cwc2 2.09 91.44 7.39 1.17 2.71 Cwc15 2.02 87.30 12.70 0.00 3.99 Ecm2 2.32 87.01 11.86 1.13 1.59 Prp45 1.77 91.19 6.74 2.07 2.40 Prp46 2.33 88.96 8.96 2.09 3.01 Cwc22 2.00 92.68 5.37 1.95 3.19 Cwc25 1.75 94.87 2.56 2.56 3.64 Yju2 2.11 92.04 6.19 1.77 4.31

10

Wan & Yan et al

Table S4 Summary of model building, refinement and validation for RNA components of the spliceosomal C complex from S. cerevisiae.

Model building software Coot & RCrane Refinement Software Phenix/Phenix.Erraser

Validation All RNAs U6 snRNA U5 snRNA U2 snRNA Pre-mRNA

Clash scores 6.32 7.89 4.59 2.65 7.85 Correct sugar puckers (%) 98.36 97.09 97.92 100.00 100.00 Good backbone (%)

73.68 69.93 77.08 83.33 68.42

Good bonds (%) 99.90 99.84 99.91 100.00 99.92 Good angles (%) 99.85 99.84 99.86 99.83 99.85

11

Wan & Yan et al

Fig. S1 A flow chart for the cryo-EM data processing and structure

determination of the spliceosomal C complex from S. cerevisiae. The final

reconstruction has an average resolution of 3.41 Å. Please refer to Materials and

Methods for details. This figure, together with Figs. S2A and S3, were prepared using

CHIMERA (67). All other structural images were created using PyMol (68).

12

Wan & Yan et al

Fig. S2 Cryo-EM analysis of a catalytic step I spliceosome (the C complex) from

Saccharomyces cerevisiae (S. cerevisiae). (A) Representative 2D class averages of

the spliceosomal C complex from S. cerevisiae. (B) The overall resolution is

estimated to be 3.41 Å on the basis of the gold standard FSC criterion of 0.143. (C)

An overall view of the EM density map for the C complex at an average resolution of

3.41 Å. The local resolutions are color-coded for different regions of the C complex.

The surface view of the spliceosome is shown here. The resolution reaches 2.9-3.5 Å

for the core regions of the C complex. (D) The EM density map is low-pass filtered

to 10 Å resolution to show the more flexible regions of the C complex.

13

Wan & Yan et al

Fig. S3 Cryo-EM analysis designed to improve the EM density maps of the

peripheral regions of the spliceosomal C complex from S. cerevisiae. Starting

from the 161,066 polished particles that yield an average resolution of 3.41 Å, we

performed two more rounds of 3D classification. After the first round of 3D

classification and auto-refinement, 80,367 particles of one major class yield a

reconstruction with an average resolution of 3.65 Å. After the second round of 3D

classification and auto-refinement, 20,686 particles give rise to a reconstruction with

an average resolutions of 4.6 Å. In both cases, the resulting EM density maps show

improved features for some regions of the C complex compared to the 3.41 Å

reconstruction.

14

Wan & Yan et al

Fig. S4 Cryo-EM analysis of the spliceosomal C complex from S. cerevisiae. (A) Angular distribution of the particles used for the reconstruction of the spliceosomal C complex at 3.65 Å resolution. Each cylinder represents one view and the height of the cylinder is proportional to the number of particles for that view. Two orientations of the C complex are shown. (B) FSC curves of the final refined model versus the overall 3.65 Å map it was refined against (black); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (green). The almost perfect overlap between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from severe overfitting. (C) Estimation of the average resolution of the cryo-EM reconstructions on the basis of the gold standard FSC criteria of 0.143. Shown here are FSC curves for the overall reconstruction of all particles for the C complex (3.41 Å, blue line, 161,066 particles) and the reconstructions designed to visualize the more flexible regions of the C complex (3.65 Å, magenta line, 80,367 particles; 4.6 Å, cyan line, 20,686 particles).

15

Wan & Yan et al

Fig. S5 EM density maps of Prp8 and Snu114 in the spliceosomal C complex.

For the central spliceosomal component Prp8, the EM density maps are shown for the

N-domain (A), RT Palm/Finger (B), Thumb/X (C), Linker (D), Endonuclease domain

(E), RNaseH-like domain (F), and five representative secondary structural elements

from these regions of Prp8 (G). For the only GTPase Snu114, the EM density maps

are shown for the overall structure (H) and three representative α-helices (I).

16

Wan & Yan et al

Fig. S6 EM density maps for the protein components of the C complex. Shown here are the density maps of the splicing factor Yju2 (A), two representative structural elements and a zinc finger of Yju2 (B), the splicing factor Cwc25 (C), the interface between Yju2 and Cwc25 (D), the NTR component Bud31 (E), a representative α-helix of Bud31 (F), the NTC component Cef1 (G), two representative secondary structural elements of Cef1 (H), the NTC component Syf2 (I), two α-helices of Syf2 (J), the NTC component Clf1 (K), and two representative α-helices of Clf1 (L).

17

Wan & Yan et al

Fig. S7 EM density maps of protein components and their interfaces with surrounding proteins. (A) The EM density maps of the NTR component Prp46 and its interface with Snu114 (residues 67-97), Prp8 (residues 737-837), Cwc15 (residues 4-41), and Prp45 (residues 30-94). Three perpendicular views are shown. (B) EM density maps at the interface among Syf2, Cef1 (residues 163-252), the N-terminal half of Clf1, and U2 snRNA. (C) EM density maps at the interface among the NTR component Prp45 (residues 156-186), the Myb domain of Cef1, Syf2, and Prp8 (residues 737-837). (D) EM density maps at the interface among the Linker domain of Prp8, MA3 domain of Cwc22, Cwc21 (residues 2-28), and 5’-exon. Two perpendicular views are shown.

18

Wan & Yan et al

Fig. S8 EM density maps of the RNA elements. (A) Overall EM density maps for the RNA elements at the center of the C complex. The four RNA molecules are color-coded. The label “5’-intron” refers to the intron sequences at the 5’-end of the intron, including the 5’-splice site (5’SS) and the ensuing nucleotides. Two perpendicular views are shown. (B) Overall EM density maps for U5 snRNA. (C) Two close-up views on the EM density maps of loop I of U5 snRNA (upper panel) and a duplex region (lower panel). (D) A close-up view on the duplex between the 5’-exon sequences (red) and loop I of U5 snRNA (orange).

19

Wan & Yan et al

Fig. S9 EM density maps of U6 snRNA and the catalytic center. (A) Overall EM density maps of U6 snRNA. (B) Two close-up views of the local EM density maps for the intramolecular stem loop (ISL) of U6 snRNA and helix II of the U2/U6 snRNA duplex. (C) Two perpendicular views of the EM density maps for the RNA elements at the catalytic center. The RNA elements include the ISL of U6 snRNA (green), loop I of U5 snRNA (orange), a small portion of U2 snRNA (marine), the 5’-exon (red), and the intron lariat (magenta).

20

Wan & Yan et al

Fig. S10 EM density maps of the active site. (A) A close-up view on the EM density maps at the active site. The two catalytic metals are shown in red spheres and the M1 magnesium ion (Mg2+) is coordinated by four ligands in a planar fashion. (B) A close-up view on the EM density maps centered around the invariant adenine nucleotide of the BPS. The 2’-OH group is covalently joined with the phosphate at the 5’-end of the 5’-splice site (5’SS). (C) A close-up view on the EM density maps centered around M1. The lariat junction and 5’-exon are shown. (D) A close-up view on the EM density maps for the lariat junction and the duplex between loop I of U5 snRNA and 5’-exon.

21

Wan & Yan et al

Fig. S11 EM density maps of the protein components around the RNA elements at the catalytic center. (A) Two overall views on the EM density maps for Yju2, Cwc25, Isy1, Syf2, and a portion of Cef1. (B) A close-up view on the EM density maps of the splicing factor Yju2. (C) A close-up view on the EM density maps of the splicing factor Cwc25. (D) A close-up view on the EM density maps of the NTC component Isy1. (E) A close-up view on the EM density maps of a portion of the NTC component Cef1. (F) A close-up view on the EM density maps of the NTC component Syf2.

22

Wan & Yan et al

Fig. S12 Structural features of the spliceosomal C complex. (A) The protein components at the periphery of the C complex. The three corners are marked by U5 Sm ring and 3’-end sequences of U5 snRNA, Msl1/Leal1/U2 Sm ring and 3’-end sequences of U2 snRNA, and the carboxyl-terminal sequences of Clf1. The RNA elements are shown to indicate the location of the catalytic center. (B) The protein components at the center of the C complex. At least 16 proteins have been identified around the catalytic center of the spliceosome. Two views are shown. Proteins shown in the left panel include Cef1, Clf1, Cwc2, Cwc15, Cwc25, Ecm2, Isy1, Prp45, Prp46, Syf2, and Yju2. Proteins shown in the right panel include Bud31, Cwc21, Cwc22, Prp8, and Snu114.

23

Wan & Yan et al

Fig. S13 Structural comparison of individual RNA elements among the three spliceosomal complexes. (A) Structural comparison of U5 snRNA from the S. cerevisiae C complex, the S. cerevisiae Bact complex (42), and the S. pombe ILS complex (10, 11). (B) Structural comparison of U6 snRNA from the three spliceosomal complexes. (C) Structural comparison of U2 snRNA from the three spliceosomal complexes. (D) Structural comparison of the lariat junctions between the S. cerevisiae C complex and S. pombe ILS complex (10, 11).

24

Wan & Yan et al

Fig. S14 The RNaseH-like domain stabilizes a mobile RNA element. (A) A close-up view on the RNaseH-like domain in the S. cerevisiae U4/U6.U5 tri-snRNP. Together with Prp3, Prp6, and Prp31, the RNaseH-like domain of Prp8 plays an important role in orienting the U4/U6 snRNA duplex. (B) A close-up view on the RNaseH-like domain in the S. cerevisiae Bact complex (42). It interacts with Hsh155 and Bud13 to stabilize placement of the intron sequences. (C) A close-up view on the RNaseH-like domain in the S. cerevisiae C complex. It interacts with Cwc25 and U2 Sm ring to stabilize placement of the intron-U2 duplex and the sequences at the 3’-end of U2 snRNA. (D) A close-up view on the RNaseH-like domain in the S. pombe ILS complex (10). It interacts with Prp19 to stabilize placement of the intron lariat.

25

References and Notes

1. S. M. Fica, N. Tuttle, T. Novak, N. S. Li, J. Lu, P. Koodathingal, Q. Dai, J. P. Staley, J. A.

Piccirilli, RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229–234 (2013).

Medline

2. M. J. Moore, C. C. Query, P. A. Sharp, Splicing of Precursors to mRNA by the Spliceosome.

The RNA World (Cold Spring Harbor Monograph Archive, 1993).

3. M. C. Wahl, C. L. Will, R. Lührmann, The spliceosome: Design principles of a dynamic RNP

machine. Cell 136, 701–718 (2009). Medline doi:10.1016/j.cell.2009.02.009

4. C. L. Will, R. Lührmann, Spliceosome structure and function. Cold Spring Harb. Perspect.

Biol. 3, a003707 (2011). Medline doi:10.1101/cshperspect.a003707

5. T. A. Steitz, J. A. Steitz, A general two-metal-ion mechanism for catalytic RNA. Proc. Natl.

Acad. Sci. U.S.A. 90, 6498–6502 (1993). Medline doi:10.1073/pnas.90.14.6498

6. E. J. Sontheimer, S. Sun, J. A. Piccirilli, Metal ion catalysis during splicing of premessenger

RNA. Nature 388, 801–805 (1997). Medline doi:10.1038/42068

7. P. M. Gordon, E. J. Sontheimer, J. A. Piccirilli, Metal ion catalysis during the exon-ligation

step of nuclear pre-mRNA splicing: Extending the parallels between the spliceosome and

group II introns. RNA 6, 199–205 (2000). Medline doi:10.1017/S1355838200992069

8. S.-L. Yean, G. Wuenschell, J. Termini, R.-J. Lin, Metal-ion coordination by U6 small nuclear

RNA contributes to catalysis in the spliceosome. Nature 408, 881–884 (2000). Medline

doi:10.1038/35048617

9. P. Koodathingal, T. Novak, J. A. Piccirilli, J. P. Staley, The DEAH box ATPases Prp16 and

Prp43 cooperate to proofread 5′ splice site cleavage during pre-mRNA splicing. Mol. Cell

39, 385–395 (2010). Medline doi:10.1016/j.molcel.2010.07.014

10. C. Yan, J. Hang, R. Wan, M. Huang, C. C. Wong, Y. Shi, Structure of a yeast spliceosome at

3.6-angstrom resolution. Science 349, 1182–1191 (2015). Medline

doi:10.1126/science.aac7629

11. J. Hang, R. Wan, C. Yan, Y. Shi, Structural basis of pre-mRNA splicing. Science 349, 1191–

1198 (2015). Medline doi:10.1126/science.aac8159

12. R. J. Grainger, J. D. Beggs, Prp8 protein: At the heart of the spliceosome. RNA 11, 533–557

(2005). Medline doi:10.1261/rna.2220705

13. W. P. Galej, C. Oubridge, A. J. Newman, K. Nagai, Crystal structure of Prp8 reveals active

site cavity of the spliceosome. Nature 493, 638–643 (2013). Medline

doi:10.1038/nature11843

14. S. Mozaffari-Jovin, T. Wandersleben, K. F. Santos, C. L. Will, R. Lührmann, M. C. Wahl,

Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8.

Science 341, 80–84 (2013). Medline doi:10.1126/science.1237515

15. T. H. Nguyen, J. Li, W. P. Galej, H. Oshikane, A. J. Newman, K. Nagai, Structural basis of

Brr2-Prp8 interactions and implications for U5 snRNP biogenesis and the spliceosome

active site. Structure 21, 910–919 (2013). Medline doi:10.1016/j.str.2013.04.017

16. A. K. Leung, K. Nagai, J. Li, Structure of the spliceosomal U4 snRNP core domain and its

implication for snRNP biogenesis. Nature 473, 536–539 (2011). Medline

doi:10.1038/nature09956

17. L. Zhou, J. Hang, Y. Zhou, R. Wan, G. Lu, P. Yin, C. Yan, Y. Shi, Crystal structures of the

Lsm complex bound to the 3′ end sequence of U6 small nuclear RNA. Nature 506, 116–

120 (2014). Medline doi:10.1038/nature12803

18. G. Weber, S. Trowitzsch, B. Kastner, R. Lührmann, M. C. Wahl, Functional organization of

the Sm core in the crystal structure of human U1 snRNP. EMBO J. 29, 4172–4184

(2010). Medline doi:10.1038/emboj.2010.295

19. D. A. Pomeranz Krummel, C. Oubridge, A. K. Leung, J. Li, K. Nagai, Crystal structure of

human spliceosomal U1 snRNP at 5.5 Å resolution. Nature 458, 475–480 (2009).

Medline doi:10.1038/nature07851

20. Y. Kondo, C. Oubridge, A. M. van Roon, K. Nagai, Crystal structure of human U1 snRNP, a

small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site

recognition. eLife 4, e04986 (2015). Medline doi:10.7554/eLife.04986

21. W. Chen, M. J. Moore, The spliceosome: Disorder and dynamics defined. Curr. Opin. Struct.

Biol. 24, 141–149 (2014). Medline doi:10.1016/j.sbi.2014.01.009

22. M. Azubel, S. G. Wolf, J. Sperling, R. Sperling, Three-dimensional structure of the native

spliceosome by cryo-electron microscopy. Mol. Cell 15, 833–839 (2004). Medline

doi:10.1016/j.molcel.2004.07.022

23. N. Behzadnia, M. M. Golas, K. Hartmuth, B. Sander, B. Kastner, J. Deckert, P. Dube, C. L.

Will, H. Urlaub, H. Stark, R. Lührmann, Composition and three-dimensional EM

structure of double affinity-purified, human prespliceosomal A complexes. EMBO J. 26,

1737–1748 (2007). Medline doi:10.1038/sj.emboj.7601631

24. S. Bessonov, M. Anokhina, A. Krasauskas, M. M. Golas, B. Sander, C. L. Will, H. Urlaub,

H. Stark, R. Lührmann, Characterization of purified human Bact

spliceosomal complexes

reveals compositional and morphological changes during spliceosome activation and first

step catalysis. RNA 16, 2384–2403 (2010). Medline doi:10.1261/rna.2456210

25. D. Boehringer, E. M. Makarov, B. Sander, O. V. Makarova, B. Kastner, R. Lührmann, H.

Stark, Three-dimensional structure of a pre-catalytic human spliceosomal complex B.

Nat. Struct. Mol. Biol. 11, 463–468 (2004). Medline doi:10.1038/nsmb761

26. P. Fabrizio, J. Dannenberg, P. Dube, B. Kastner, H. Stark, H. Urlaub, R. Lührmann, The

evolutionarily conserved core design of the catalytic activation step of the yeast

spliceosome. Mol. Cell 36, 593–608 (2009). Medline doi:10.1016/j.molcel.2009.09.040

27. M. M. Golas, B. Sander, S. Bessonov, M. Grote, E. Wolf, B. Kastner, H. Stark, R.

Lührmann, 3D cryo-EM structure of an active step I spliceosome and localization of its

catalytic core. Mol. Cell 40, 927–938 (2010). Medline doi:10.1016/j.molcel.2010.11.023

28. M. Grote, E. Wolf, C. L. Will, I. Lemm, D. E. Agafonov, A. Schomburg, W. Fischle, H.

Urlaub, R. Lührmann, Molecular architecture of the human Prp19/CDC5L complex. Mol.

Cell. Biol. 30, 2105–2119 (2010). Medline doi:10.1128/MCB.01505-09

29. M. S. Jurica, D. Sousa, M. J. Moore, N. Grigorieff, Three-dimensional structure of C

complex spliceosomes by electron microscopy. Nat. Struct. Mol. Biol. 11, 265–269

(2004). Medline doi:10.1038/nsmb728

30. M. D. Ohi, L. Ren, J. S. Wall, K. L. Gould, T. Walz, Structural characterization of the fission

yeast U5.U2/U6 spliceosome complex. Proc. Natl. Acad. Sci. U.S.A. 104, 3195–3200

(2007). Medline doi:10.1073/pnas.0611591104

31. B. Sander, M. M. Golas, E. M. Makarov, H. Brahms, B. Kastner, R. Lührmann, H. Stark,

Organization of core spliceosomal components U5 snRNA loop I and U4/U6 di-snRNP

within U4/U6.U5 tri-snRNP as revealed by electron cryomicroscopy. Mol. Cell 24, 267–

278 (2006). Medline doi:10.1016/j.molcel.2006.08.021

32. E. Wolf, B. Kastner, J. Deckert, C. Merz, H. Stark, R. Lührmann, Exon, intron and splice site

locations in the spliceosomal B complex. EMBO J. 28, 2283–2292 (2009). Medline

doi:10.1038/emboj.2009.171

33. E. M. Makarov, O. V. Makarova, H. Urlaub, M. Gentzel, C. L. Will, M. Wilm, R. Lührmann,

Small nuclear ribonucleoprotein remodeling during catalytic activation of the

spliceosome. Science 298, 2205–2208 (2002). Medline doi:10.1126/science.1077783

34. J. Deckert, K. Hartmuth, D. Boehringer, N. Behzadnia, C. L. Will, B. Kastner, H. Stark, H.

Urlaub, R. Lührmann, Protein composition and electron microscopy structure of affinity-

purified human spliceosomal B complexes isolated under physiological conditions. Mol.

Cell. Biol. 26, 5528–5543 (2006). Medline doi:10.1128/MCB.00582-06

35. M. S. Jurica, L. J. Licklider, S. R. Gygi, N. Grigorieff, M. J. Moore, Purification and

characterization of native spliceosomes suitable for three-dimensional structural analysis.

RNA 8, 426–439 (2002). Medline doi:10.1017/S1355838202021088

36. J. O. Ilagan, R. J. Chalkley, A. L. Burlingame, M. S. Jurica, Rearrangements within human

spliceosomes captured after exon ligation. RNA 19, 400–412 (2013). Medline

doi:10.1261/rna.034223.112

37. W. Chen, H. P. Shulha, A. Ashar-Patel, J. Yan, K. M. Green, C. C. Query, N. Rhind, Z.

Weng, M. J. Moore, Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron

lariat spliceosomes. RNA 20, 308–320 (2014). Medline doi:10.1261/rna.040980.113

38. T. H. Nguyen, W. P. Galej, X. C. Bai, C. G. Savva, A. J. Newman, S. H. Scheres, K. Nagai,

The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47–52 (2015).

Medline doi:10.1038/nature14548

39. D. E. Agafonov, B. Kastner, O. Dybkov, R. V. Hofele, W. T. Liu, H. Urlaub, R. Lührmann,

H. Stark, Molecular architecture of the human U4/U6.U5 tri-snRNP. Science 351, 1416–

1420 (2016). Medline doi:10.1126/science.aad2085

40. R. Wan, C. Yan, R. Bai, L. Wang, M. Huang, C. C. Wong, Y. Shi, The 3.8 Å structure of the

U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis. Science 351,

466–475 (2016). Medline doi:10.1126/science.aad6466

41. T. H. Nguyen, W. P. Galej, X. C. Bai, C. Oubridge, A. J. Newman, S. H. Scheres, K. Nagai,

Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530,

298–302 (2016). Medline doi:10.1038/nature16940

42. C. Yan, R. Wan, R. Bai, G. Huang, Y. Shi, Structure of a yeast activated spliceosome at 3.5

Å resolution. Science 10.1126/science.aag0291 (2016). 10.1126/science.aag0291

43. A. H. Igel, M. Ares Jr., Internal sequences that distinguish yeast from metazoan U2 snRNA

are unnecessary for pre-mRNA splicing. Nature 334, 450–453 (1988). Medline

doi:10.1038/334450a0

44. A. J. Newman, C. Norman, U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites.

Cell 68, 743–754 (1992). Medline doi:10.1016/0092-8674(92)90149-7

45. J. R. Wyatt, E. J. Sontheimer, J. A. Steitz, Site-specific cross-linking of mammalian U5

snRNP to the 5′ splice site before the first step of pre-mRNA splicing. Genes Dev. 6,

2542–2553 (1992). Medline doi:10.1101/gad.6.12b.2542

46. E. J. Sontheimer, J. A. Steitz, The U5 and U6 small nuclear RNAs as active site components

of the spliceosome. Science 262, 1989–1996 (1993). Medline

doi:10.1126/science.8266094

47. A. J. Newman, S. Teigelkamp, J. D. Beggs, snRNA interactions at 5′ and 3′ splice sites

monitored by photoactivated crosslinking in yeast spliceosomes. RNA 1, 968–980 (1995).

Medline

48. M. Anokhina, S. Bessonov, Z. Miao, E. Westhof, K. Hartmuth, R. Lührmann, RNA structure

analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the

catalytic core. EMBO J. 32, 2804–2818 (2013). Medline doi:10.1038/emboj.2013.198

49. H. D. Madhani, C. Guthrie, A novel base-pairing interaction between U2 and U6 snRNAs

suggests a mechanism for the catalytic activation of the spliceosome. Cell 71, 803–817

(1992). Medline doi:10.1016/0092-8674(92)90556-R

50. C. K. Tseng, S. C. Cheng, Both catalytic steps of nuclear pre-mRNA splicing are reversible.

Science 320, 1782–1784 (2008). Medline doi:10.1126/science.1158993

51. C. G. Burns, R. Ohi, A. R. Krainer, K. L. Gould, Evidence that Myb-related CDC5 proteins

are required for pre-mRNA splicing. Proc. Natl. Acad. Sci. U.S.A. 96, 13789–13794

(1999). Medline doi:10.1073/pnas.96.24.13789

52. W. H. McDonald, R. Ohi, N. Smelkova, D. Frendewey, K. L. Gould, Myb-related fission

yeast cdc5p is a component of a 40S snRNP-containing complex and is essential for pre-

mRNA splicing. Mol. Cell. Biol. 19, 5352–5362 (1999). Medline

doi:10.1128/MCB.19.8.5352

53. T. Villa, C. Guthrie, The Isy1p component of the NineTeen complex interacts with the

ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev. 19, 1894–

1904 (2005). Medline doi:10.1101/gad.1336305

54. C. H. Chen, W. C. Yu, T. Y. Tsao, L. Y. Wang, H. R. Chen, J. Y. Lin, W. Y. Tsai, S. C.

Cheng, Functional and physical interactions between components of the Prp19p-

associated complex. Nucleic Acids Res. 30, 1029–1037 (2002). Medline

doi:10.1093/nar/30.4.1029

55. J. C. McGrail, A. Krause, R. T. O’Keefe, The RNA binding protein Cwc2 interacts directly

with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA

splicing. Nucleic Acids Res. 37, 4205–4217 (2009). Medline doi:10.1093/nar/gkp341

56. N. Rasche, O. Dybkov, J. Schmitzová, B. Akyildiz, P. Fabrizio, R. Lührmann, Cwc2 and its

human homologue RBM22 promote an active conformation of the spliceosome catalytic

centre. EMBO J. 31, 1591–1604 (2012). Medline doi:10.1038/emboj.2011.502

57. D. Xu, J. D. Friesen, Splicing factor slt11p and its involvement in formation of U2/U6 helix

II in activation of the yeast spliceosome. Mol. Cell. Biol. 21, 1011–1023 (2001). Medline

doi:10.1128/MCB.21.4.1011-1023.2001

58. D. Saha, P. Khandelia, R. T. O’Keefe, U. Vijayraghavan, Saccharomyces cerevisiae

NineTeen complex (NTC)-associated factor Bud31/Ycr063w assembles on precatalytic

spliceosomes and improves first and second step pre-mRNA splicing efficiency. J. Biol.

Chem. 287, 5390–5399 (2012). Medline doi:10.1074/jbc.M111.298547

59. R. J. Grainger, J. D. Barrass, A. Jacquier, J. C. Rain, J. D. Beggs, Physical and genetic

interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at

the catalytic center of the spliceosome. RNA 15, 2161–2173 (2009). Medline

doi:10.1261/rna.1908309

60. M. Khanna, H. Van Bakel, X. Tang, J. A. Calarco, T. Babak, G. Guo, A. Emili, J. F.

Greenblatt, T. R. Hughes, N. J. Krogan, B. J. Blencowe, A systematic characterization of

Cwc21, the yeast ortholog of the human spliceosomal protein SRm300. RNA 15, 2174–

2185 (2009). Medline doi:10.1261/rna.1790509

61. Y. C. Liu, H. C. Chen, N. Y. Wu, S. C. Cheng, A novel splicing factor, Yju2, is associated

with NTC and acts after Prp2 in promoting the first catalytic reaction of pre-mRNA

splicing. Mol. Cell. Biol. 27, 5403–5413 (2007). Medline doi:10.1128/MCB.00346-07

62. T. W. Chiang, S. C. Cheng, A weak spliceosome-binding domain of Yju2 functions in the

first step and bypasses Prp16 in the second step of splicing. Mol. Cell. Biol. 33, 1746–

1755 (2013). Medline doi:10.1128/MCB.00035-13

63. Y. F. Chiu, Y. C. Liu, T. W. Chiang, T. C. Yeh, C. K. Tseng, N. Y. Wu, S. C. Cheng, Cwc25

is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic

reaction. Mol. Cell. Biol. 29, 5671–5678 (2009). Medline doi:10.1128/MCB.00773-09

64. Z. Warkocki, P. Odenwälder, J. Schmitzová, F. Platzmann, H. Stark, H. Urlaub, R. Ficner, P.

Fabrizio, R. Lührmann, Reconstitution of both steps of Saccharomyces cerevisiae

splicing with purified spliceosomal components. Nat. Struct. Mol. Biol. 16, 1237–1243

(2009). Medline doi:10.1038/nsmb.1729

65. M. A. Wall, D. E. Coleman, E. Lee, J. A. Iñiguez-Lluhi, B. A. Posner, A. G. Gilman, S. R.

Sprang, The structure of the G protein heterotrimer Giα1β1γ2. Cell 83, 1047–1058 (1995).

Medline doi:10.1016/0092-8674(95)90220-1

66. T. A. Cooper, L. Wan, G. Dreyfuss, RNA and disease. Cell 136, 777–793 (2009). Medline

doi:10.1016/j.cell.2009.02.011

67. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T.

E. Ferrin, UCSF Chimera—A visualization system for exploratory research and analysis.

J. Comput. Chem. 25, 1605–1612 (2004). Medline doi:10.1002/jcc.20084

68. W. L. DeLano, The PyMOL Molecular Graphics System (2002); www.pymol.org.

69. S. H. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure

determination. J. Struct. Biol. 180, 519–530 (2012). Medline

doi:10.1016/j.jsb.2012.09.006

70. S. H. Scheres, Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife

3, e03665 (2014). Medline doi:10.7554/eLife.03665

71. S. Chen, G. McMullan, A. R. Faruqi, G. N. Murshudov, J. M. Short, S. H. Scheres, R.

Henderson, High-resolution noise substitution to measure overfitting and validate

resolution in 3D structure determination by single particle electron cryomicroscopy.

Ultramicroscopy 135, 24–35 (2013). Medline doi:10.1016/j.ultramic.2013.06.004

72. P. B. Rosenthal, R. Henderson, Optimal determination of particle orientation, absolute hand,

and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745

(2003). Medline doi:10.1016/j.jmb.2003.07.013

73. A. Kucukelbir, F. J. Sigworth, H. D. Tagare, Quantifying the local resolution of cryo-EM

density maps. Nat. Methods 11, 63–65 (2014). Medline doi:10.1038/nmeth.2727

74. P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr.

D 60, 2126–2132 (2004). Medline doi:10.1107/S0907444904019158

75. G. N. Murshudov, A. A. Vagin, E. J. Dodson, Refinement of macromolecular structures by

the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997). Medline

doi:10.1107/S0907444996012255

76. K. S. Keating, A. M. Pyle, RCrane: Semi-automated RNA model building. Acta Crystallogr.

D 68, 985–995 (2012). Medline doi:10.1107/S0907444912018549

77. F. C. Chou, P. Sripakdeevong, S. M. Dibrov, T. Hermann, R. Das, Correcting pervasive

errors in RNA crystallography through enumerative structure prediction. Nat. Methods

10, 74–76 (2013). Medline doi:10.1038/nmeth.2262

78. M. M. Harding, Geometry of metal-ligand interactions in proteins. Acta Crystallogr. D 57,

401–411 (2001). Medline doi:10.1107/S0907444900019168

79. M. M. Harding, Metal-ligand geometry relevant to proteins and in proteins: Sodium and

potassium. Acta Crystallogr. D 58, 872–874 (2002). Medline

doi:10.1107/S0907444902003712

80. M. C. Erat, R. K. Sigel, Divalent metal ions tune the self-splicing reaction of the yeast

mitochondrial group II intron Sc.ai5γ. J. Biol. Inorg. Chem. 13, 1025–1036 (2008).

Medline doi:10.1007/s00775-008-0390-7

81. P. Auffinger, L. Bielecki, E. Westhof, Anion binding to nucleic acids. Structure 12, 379–388

(2004). Medline doi:10.1016/j.str.2004.02.015

82. M. Marcia, A. M. Pyle, Principles of ion recognition in RNA: Insights from the group II

intron structures. RNA 20, 516–527 (2014). Medline doi:10.1261/rna.043414.113

83. J. Mähler, I. Persson, A study of the hydration of the alkali metal ions in aqueous solution.

Inorg. Chem. 51, 425–438 (2012). Medline doi:10.1021/ic2018693

84. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd,

L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R.

Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart,

PHENIX: A comprehensive Python-based system for macromolecular structure solution.

Acta Crystallogr. D 66, 213–221 (2010). Medline doi:10.1107/S0907444909052925

85. R. A. Nicholls, M. Fischer, S. McNicholas, G. N. Murshudov, Conformation-independent

structural comparison of macromolecules with ProSMART. Acta Crystallogr. D 70,

2487–2499 (2014). Medline doi:10.1107/S1399004714016241

86. A. Amunts, A. Brown, X. C. Bai, J. L. Llácer, T. Hussain, P. Emsley, F. Long, G.

Murshudov, S. H. Scheres, V. Ramakrishnan, Structure of the yeast mitochondrial large

ribosomal subunit. Science 343, 1485–1489 (2014). Medline

doi:10.1126/science.1249410

87. I. W. Davis, A. Leaver-Fay, V. B. Chen, J. N. Block, G. J. Kapral, X. Wang, L. W. Murray,

W. B. Arendall III, J. Snoeyink, J. S. Richardson, D. C. Richardson, MolProbity: All-

atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res.

35 (suppl. 2), W375–W383 (2007). Medline doi:10.1093/nar/gkm216

88. B. A. Barad, N. Echols, R. Y. Wang, Y. Cheng, F. DiMaio, P. D. Adams, J. S. Fraser,

EMRinger: Side chain-directed model and map validation for 3D cryo-electron

microscopy. Nat. Methods 12, 943–946 (2015). Medline doi:10.1038/nmeth.3541