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
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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 radiationdamage weighting (known as particle polishing) (70), these polished particles give a
reconstruction with an improved average resolution of 3.41 Å following autorefinement (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
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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).
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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.
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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
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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 wellrefined 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.
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Table S1. Cryo-EM data collection and refinement statistics.
Data collection
EM equipment
Voltage (kV)
Detector
Pixel size (Å)
Electron dose (e-/Å2)
Defocus range (µm)
Reconstruction
Software
Number of used Particles
Accuracy of rotation (˚)
Accuracy of translation (pixels)
Final Resolution (Å)
Model building
software
Refinement
Software
Map sharpening B-factor (Å2)
Average Fourier shell correlation
R-factor
Model composition
Protein residues
RNA nucleotides
GTP
Validation
R.m.s deviations
Bonds length (Å)
Bonds Angle (˚)
Ramachandran plot statistics (％)
Preferred
Allowed
Outlier
FEI Titan Krios
300
Gatan K2
1.306
45.6
1.6~2.6
RELION 1.4
161,066 / 80,367
0.441 / 0.494
0.402 / 0.405
3.41 / 3.65
Coot & Rosetta
Phenix & Refmac
-83.4
0.893
0.245
8,587
434
1
0.010
1.343
92.48
5.94
1.59
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Table S2 Summary of model building for the yeast spliceosomal C complex.
U5 snRNP
U6 snRNP
U2 snRNP
NTC/Prp19
Complex
NTC-Related
proteins
Known
Splicing
Factors
Step2
proteins
Pre-mRNA
Molecule
U5 snRNA
Length
214
Prp8
2413
Snu114
SmB1
SmD1
SmD2
SmD3
SmE1
SmF1
SmG1
U6 snRNA
U2 RNA
984
196
146
110
101
94
86
77
112 nt
1175 nt
Lea1
Msl1
SmB1
SmD1
SmD2
SmD3
SmE1
SmF1
SmG1
Clf1
238
111
196
146
110
101
94
86
77
687
Domain/Region
28:183
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)
67:975
Sm fold
Sm fold
Sm fold
Sm fold
Sm fold
Sm fold
Sm fold
1:106
1:48
53:107
109:191
LRR domain
RRM domain
Sm fold
Sm fold
Sm fold
Sm fold
Sm fold
Sm fold
Sm fold
TPR domain (40:275)
-
PDB code
From Bact
Modeling
Homology modeling
Resolution (Å)
2.9~4.0
From Bact
Homology modeling
2.9~4.0
From Bact
Not modeled
Homology modeling
~4.0
2.9~4.0
From Bact
Rigid docking
From Bact
From Bact
3JB9
3JB9
3JB9
Homology modeling
Homology modeling
RNA duplex
Rigid Docking
Rigid Docking
Rigid Docking
3JB9
Rigid docking
From Bact
3JB9
3JB9
Homology modeling
Rigid docking
Rigid docking
3.0~4.5
~30
~30
From Bact
3JB9
3JB9
3JB9
Homology modeling
Homology modeling
Rigid docking
Rigid docking
3.0~3.5
3.0~3.5
~30
~30
4.0~6.0
2.9~4.0
2.9~4.0
5.0~8.0
5.0~8.0
5.0~8.0
5.0~8.0
5.0~8.0
Syf1
859
Cef1
590
Prp19
503
Myb Domain (9:111)
145:253
-
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
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
Cwc21
Cwc22
135
577
From Bact
From Bact
Cwc25
Yju2
Prp17
179
278
455
2:28
MIF4G domain (11:263)
MA3 domain (279:485)
N-terminal domain (2:42)
2:116
50:75
3JB9
Homology modeling
Rigid docking
Homology modeling
De novo building
De novo building
Homology modeling
3.0~4.0
4~10
3.0~3.5
2.8~3.2
3.0~4.0
3.0~4.2
Pre-mRNA
-
57nt
From Bact
De novo building
2.9~4.5
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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
Molecule
Molprobity
Scores
Ramachandran plot statistics (％)
EMRinger*
Score
Preferred
Allowed
Outlier
Prp8
2.16
3.13
92.44
6.04
1.52
Snu114
2.19
2.97
91.86
6.88
1.26
Clf1
2.24
2.57
93.28
6.72
0.00
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
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.
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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
Clash scores
Correct sugar puckers (%)
Good
backbone
(%)
Good bonds (%)
Good angles (%)
All RNAs
U6 snRNA
U5 snRNA
U2 snRNA
Pre-mRNA
6.32
98.36
73.68
99.90
99.85
7.89
97.09
69.93
99.84
99.84
4.59
97.92
77.08
99.91
99.86
2.65
100.00
83.33
100.00
99.83
7.85
100.00
68.42
99.92
99.85
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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).
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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.
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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.
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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).
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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).
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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 αhelix 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).
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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.
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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 colorcoded. 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).
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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).
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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.
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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.
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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.
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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).
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Fig. S14 The RNaseH-like domain stabilizes a mobile RNA element. (A) A closeup 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.
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2. M. J. Moore, C. C. Query, P. A. Sharp, Splicing of Precursors to mRNA by the Spliceosome.
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