Research Article

Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation

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Science  29 Jun 2018:
Vol. 360, Issue 6396, pp. 1423-1429
DOI: 10.1126/science.aau0325

Structural basis for spliceosome assembly

The spliceosome removes noncoding sequences from precursor RNA and ligates coding sequences into useful mRNA. The pre-spliceosome (A complex) associates with a small nuclear ribonucleoprotein (snRNP) complex called U4/U6.U5 tri-snRNP to form the pre-B complex, which is converted into the precatalytic B complex. Bai et al. solved the cryo–electron microscopy structures of the pre-B and B complexes isolated from yeast. These structures show the U1 and U2 snRNPs and allow modeling of the A complex to reveal the early steps of spliceosome assembly and activation.

Science, this issue p. 1423

Abstract

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo–electron microscopy structures of the Saccharomyces cerevisiae pre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5′ splice site (5′SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5′-exon–5′SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

Assembly and activation of the spliceosome take place in an ordered process (13). First, the 5′ splice site (5′SS) and the branch point sequence (BPS) are recognized by the U1 and U2 small nuclear ribonucleoproteins (snRNPs), respectively, through duplex formation with U1 and U2 snRNAs in the pre-spliceosome (known as the A complex). Then, the A complex associates with the U4/U6.U5 tri-snRNP to form the pre-B complex, the first fully assembled spliceosome that contains all five snRNPs (4). The adenosine triphosphatase (ATPase)/helicase Prp28 drives the dissociation of U1 snRNP, freeing the 5′SS and 5′ exon for recognition by the U6 and U5 small nuclear RNAs (snRNAs), respectively (57). The resulting B complex is converted by the ATPase/helicase Brr2 into the activated spliceosome (Bact complex). The Bact complex is remodeled to become the catalytically activated spliceosome (B* complex), where the branching reaction occurs. The resulting catalytic step I spliceosome (C complex) is converted into the step II catalytically activated spliceosome (C* complex), and exon ligation follows. The ligated exon in the postcatalytic spliceosome (P complex) is released, and the resulting intron lariat spliceosome (ILS) is disassembled, completing one cycle of precursor messenger RNA (pre-mRNA) splicing.

Structure elucidation of the yeast spliceosome has led to major advances in the mechanistic understanding of pre-mRNA splicing (810). Since determination of the 3.6-Å structure of the Schizosaccharomyces pombe ILS complex in 2015 (11, 12), cryo–electron microscopy (cryo-EM) structures at atomic or near-atomic resolutions have been reported for the Saccharomyces cerevisiae B, Bact, C, C*, P, and ILS complexes. Here we report the cryo-EM structure of the S. cerevisiae pre-B complex at resolutions of 3.3, 3.6 to 4.6, and 3.4 Å for U1 snRNP, U2 snRNP, and the tri-snRNP, respectively. We also report the structure of the S. cerevisiae B complex at 3.9-Å resolution.

Electron microscopy of the endogenous pre-B complex

The endogenous pre-B and B complexes were individually derived from two different strains of S. cerevisiae. In both cases, the spliceosome was purified through two steps of affinity chromatography (fig. S1, A and B), and its identity was confirmed by snRNA analysis (fig. S1, C and D). Chemical cross-linking was used to stabilize the otherwise highly dynamic pre-B and B complexes. To overcome the transient nature of the pre-B complex, we engineered a mutant Prp28 that blocks the dissociation of U1 snRNP (13). Cryo-EM samples were imaged by a K2 Summit detector (Gatan) mounted on a Titan Krios electron microscope (FEI) (fig. S1, E and F).

Low-resolution references of the pre-B and B complexes were derived from a preliminary analysis of the EM data (fig. S2). For the S. cerevisiae pre-B complex, 1.85 million particles were autopicked and classified using a guided multireference procedure, as reported previously (14) (fig. S3). Owing to the motions of U1 and U2 snRNPs relative to the tri-snRNP, subsequent three-dimensional classifications were applied with local masks. Structures of U1 snRNP, U2 snRNP, and tri-snRNP were determined at average resolutions of 3.3, 3.6 to 4.6, and 3.4 Å, respectively (fig. S3 and table S1). A similar procedure yielded a reconstruction of the B complex at an average resolution of 3.9 Å (figs. S4 and S5A and table S1). The local resolution reaches 3.0 Å in the core regions of the yeast pre-B and B complexes (fig. S5B), with valid EM analysis (fig. S5, C to F). The EM maps exhibit distinguishing features of nucleotides and amino acid side chains (fig. S6). Atomic modeling of the pre-B and B complexes was aided by the structures of the yeast U1 snRNP (15), the U4/U6.U5 tri-snRNP (16, 17), and the B (18) and Bact (19) complexes (tables S2 and S3).

Structure of the pre-B complex

The structure of the S. cerevisiae pre-B complex contains 68 discrete proteins, five snRNA molecules, and the pre-mRNA (Fig. 1A). The structurally identified proteins include 16 in U1 snRNP (Luc7, Mud1/U1-A, Nam8, Prp39, Prp42, Snp1/U1-70K, Snu56, Snu71, Yhc1/U1-C, and the U1 Sm ring), 18 in U2 snRNP, 31 in the U4/U6.U5 tri-snRNP, and 3 in the RES complex (Bud13, Pml1, and Snu17). The protein components of U2 snRNP are distributed in the SF3a complex (Prp9, Prp11, and Prp21), the SF3b complex (Cus1, Hsh49, Hsh155, Rse1, Rds3, and Ysf3), and the U2 core (Lea1, Msl1, and the U2 Sm ring). Proteins of the tri-snRNP include 11 in U4 snRNP (Prp3, Prp4, Prp31, Snu13, and the U4 Sm ring), 11 in U5 snRNP (Brr2, Dib1, Prp8, Snu114, and the U5 Sm ring), the U6 LSm ring, and 2 tri-snRNP–specific proteins (Prp6 and Snu66).

Fig. 1 Cryo-EM structures of the pre-B and B complexes from S. cerevisiae.

(A) Structure of the pre-B complex. The pre-B complex comprises U1 snRNP, U2 snRNP, and the U4/U6.U5 tri-snRNP. U2 snRNP comprises three subcomplexes: the SF3a complex, the SF3b complex, and the U2 core. The pre-mRNA and the U1, U2, U4, U5, and U6 snRNAs are colored red, yellow, marine, violet, orange, and green, respectively. (B) Structures of the B complex. The coloring scheme is the same as in (A) except that the B-specific proteins (Prp38, Spp381, and Snu23) are included. All structural images were created using PyMol (41).

U1 snRNP is relatively compact and well defined by the 3.3-Å EM map (fig. S6). U2 snRNP has an elongated shape and exhibits considerable internal flexibility; SF3a bridges SF3b and the U2 core (fig. S7). The pre-mRNA retention and splicing (RES) complex binds Hsh155 and the 3′-end sequences of the intron (fig. S7, A and C). The tri-snRNP is well characterized by the EM map (fig. S8). The structures and locations of most protein and RNA components in the tri-snRNP are nearly identical to those in the isolated yeast tri-snRNP (16, 17). The U1 and U2 snRNPs loosely interact with each other (fig. S7, A and B), and they only make limited contacts with the tri-snRNP. Consequently, the entire pre-B complex exhibits considerable flexibility, with five rigid parts (U1 snRNP, SF3a, SF3b, U2 core, and tri-snRNP) loosely bound together to generate a highly asymmetric assembly (Fig. 1A).

The structure of the S. cerevisiae B complex contains 55 proteins, 4 snRNA molecules, and the pre-mRNA (Fig. 1B). Compared with the pre-B complex, Prp38, Snu23, and Spp381 are recruited further into the B complex (2023). These proteins form a subcomplex and appear to orient the pre-mRNA and facilitate the recognition of pre-mRNA by U6 snRNA. Except for Brr2, the U4 Sm ring, and the RNaseH-like and Jab1/MPN domains of Prp8, all other proteins in the tri-snRNP of the B complex remain structurally identical to those in the pre-B complex. U2 snRNP appears to collapse onto the tri-snRNP in the B complex, forging closer interactions than in the pre-B complex (Fig. 1B).

RNA elements in the pre-B and B complexes

In the pre-B complex, the 5′SS and BPS are recognized by the U1 and U2 snRNPs, respectively, in part through duplex formation with U1 and U2 snRNAs (Fig. 2, A and B). Eleven consecutive nucleotides (A1UACUUACCUU11) at the 5′ end of U1 snRNA base-pair with the 5′SS (GUAUGU) and its surrounding nucleotides (Fig. 2C). Fifteen consecutive nucleotides (G32UGUAGUAUCUGUUC46) of U2 snRNA form a duplex with the BPS (UACUAAC) and its surrounding nucleotides (Fig. 2D). The 5′-end sequences of U2 snRNA already form helix II with the 3′-end sequences of U6 snRNA, and five consecutive nucleotides of U6 snRNA (A26U27U28U29G30) are anchored to loop I of U5 snRNA through duplex formation (Fig. 2E and fig. S8D).

Fig. 2 The RNA elements in the S. cerevisiae pre-B and B complexes.

(A) Structural comparison of the RNA elements of the pre-B and B complexes. The RNA elements in the pre-B complex are colored identically to those in Fig. 1A, whereas in the B complex, the pre-mRNA is colored wheat and all other RNA elements are shown in gray. (B) Schematic diagrams of the base-pairing interactions among the RNA elements in the pre-B complex (left panel) and the B complex (right panel). (C) A close-up view of the RNA duplex between the 5′SS and the complementary U1 snRNA sequences in the pre-B complex. (D) A close-up view of the RNA duplex between the BPS and the complementary U2 snRNA sequences in the pre-B complex. The nucleophile-containing adenine base is already flipped out of the duplex registry. (E) A close-up view of the duplex between U6 snRNA and loop I of U5 snRNA in the pre-B complex. Five consecutive nucleotides (A26U27U28U29G30) of U6 snRNA form a duplex with U96U97U98U99A100 of U5 loop I. (F) A close-up view of the interactions between the 5′-exon–5′SS sequences and U6 snRNA in the B complex. The 5′-exon–5′SS sequences are located close to U6 snRNA.

U1 snRNP is absent in the B complex. The freed 5′-exon–5′SS sequences are translocated by ~90 Å (relative to their position in the pre-B complex) to the vicinity of the ACAGA box of U6 snRNA (Fig. 2B). Three nucleotides of the pre-mRNA interact with the tip of the ACAGA stem loop of U6 snRNA (Fig. 2F). However, the 5′SS and 5′ exon are yet to be recognized by the ACAGA box and U5 loop I, respectively. The majority of the snRNA elements remain unchanged in the transition from pre-B to B. These include U5 snRNA in its entirety; U6 snRNA, except 20 nucleotides at the 3′ end; and the bulk of U4 snRNA (Fig. 2, A and B). The 5′ portion of U2 snRNA, as helix II of the U2/U6 duplex, undergoes a slight positional shift in the transition. The only snRNA element that exhibits marked changes is the middle portion of U2 snRNA.

Recognition of the 5′SS by U1 snRNP

The cryo-EM structure of the S. cerevisiae U1 snRNP was previously determined in the absence of the pre-mRNA (15) (fig. S9A). Our structure of the pre-B complex reveals how the 5′SS is recognized by U1 snRNP in an intact spliceosome (Fig. 3 and fig. S9A). The 5′SS and U1 snRNA form an extended duplex, which is recognized by Yhc1, Luc7, and SmB and SmD3 of the U1 Sm ring (Fig. 3A). The positive electrostatic surface potential of these proteins may neutralize the negative charges of the 5′SS/U1 duplex (Fig. 3B). These general structural features corroborate the reported biochemical functions of Yhc1 and Luc7 (2426). Yhc1 (U1-C in humans) is an essential subunit of U1 snRNP and plays an important role in stabilizing the 5′SS/U1 duplex (24, 2729).

Fig. 3 Recognition of the 5′SS by U1 snRNP in the S. cerevisiae pre-B complex.

(A) The 5′SS/U1 snRNA duplex directly interacts with Yhc1, Luc7, and the U1 Sm ring. UNK, unknown protein. (B) The acidic 5′SS/U1 snRNA duplex is likely stabilized by the positive charges on the surface of the surrounding proteins. The protein components are shown by their electrostatic surface potential. (C) Two close-up views of the specific interactions between the 5′SS/U1 duplex and residues from Yhc1. (D) Sequence alignment of Yhc1 (S. cerevisiae) with its orthologs Usp103 (S. pombe) and U1-C (H. sapiens). Conserved residues are boxed, and invariant residues are shaded red. Residues that may mediate H-bonds are marked by black arrows. (E) A close-up view of the interactions between the 5′SS/U1 duplex and residues from Luc7. (F) Sequence alignment of Luc7 (S. cerevisiae) with its orthologs Usp106 (S. pombe) and Luc7L (H. sapiens). (G) A close-up view of the interactions between the 5′SS/U1 duplex and residues from SmB1 and SmD3 of the U1 Sm ring. (H) A summary of the interactions between 5′SS/U1 and surrounding proteins. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Yhc1 contributes a number of hydrogen bonds (H-bonds) to the phosphodiester backbone of the 5′SS/U1 duplex (Fig. 3C and fig. S9, B and C). Three residues in the N-terminal C2H2-type zinc finger of Yhc1 (His15, Thr17, and Ser19) each donate a H-bond to the U1 snRNA strand of the 5′SS/U1 duplex (Fig. 3C, left panel); Lys28 and Asn29 make H-bonds to the pre-mRNA strand of the duplex (Fig. 3C, right panel). These residues are generally conserved in the S. pombe and human orthologs (Fig. 3D). Consistent with the crystal structure of the human U1 snRNP (27), our structural findings explain the observation that mutations of Ser19 and Val20 compromise the ability of Yhc1 to stabilize the 5′SS/U1 duplex (25).

A C-terminal fragment (residues 198 to 230) of Luc7 also forms a C2H2-type zinc finger (fig. S9, B and C), which is known to promote the splicing of pre-mRNA with a weak 5′SS (26, 30, 31). In our structure, three charged residues from this zinc finger—Asp212, Arg216, and Lys224—directly contact the 5′SS/U1 duplex through H-bonds (Fig. 3E). These three residues are invariant in the Luc7 orthologs Usp106 (S. pombe) and Luc7L (Homo sapiens) (Fig. 3F). In addition, the C-terminal residues of SmB and SmD3 interact with the 5′SS/U1 duplex (Fig. 3G). Together, these specific H-bonds, mostly made to the backbone of the 5′SS/U1 duplex, contribute to its specific accommodation by U1 snRNP (Fig. 3H).

Structure of U2 snRNP

In the published cryo-EM structure of the S. cerevisiae B complex (18), the local resolution around U2 snRNP was estimated to be 17.2 Å, which allowed docking of known structures. The improved local resolution (3.6 to 4.6 Å) around U2 snRNP in our structure of the pre-B complex offers considerably more structural details (Fig. 4A). The SF3b complex is connected to the U2 core by the SF3a complex. Within the SF3a complex (32), Prp11 binds the SF3b complex, Prp9 interacts with the U2 core, and Prp21 connects Prp11 and Prp9. Specifically, the helices α7 and α9 from Prp9 associate with SmD1 and SmD2 of the U2 Sm ring (Fig. 4B). The N-terminal 90 residues (residues 15 to 105) of Prp11 form a folded domain that is stabilized by a C2H2-coordinated (Cys68, Cys71, His84, and His90) zinc ion. The N-terminal fragment of Prp11 (residues 15 to 66) closely interacts with Cus1 and Hsh155, whereas the zinc-binding motif directly binds the BPS/U2 duplex (Fig. 4C).

Fig. 4 Structure of U2 snRNP in the S. cerevisiae pre-B complex.

(A) Overall structure of U2 snRNP in the pre-B complex. The protein components in the SF3b complex and the U2 core are colored purple and cyan, respectively. The three proteins of the SF3a complexes—Prp11, Prp21, and Prp9—are colored green, forest green, and pale green, respectively. (B) A close-up view of the interface between Prp9 of the SF3a complex and SmD1 and SmD2 of the U2 Sm ring. (C) A close-up view of the interface between Prp11 of the SF3a complex and Cus1 and Hsh155 of the SF3b complex. H15, H16, and H17 are helices.

The transition from pre-B to B

In the pre-B complex, U1 snRNP, U2 snRNP, and the U4/U6.U5 tri-snRNP interact with each other through three loose interfaces, which yield considerable flexibility (Fig. 5A). At the interface between the U1 and U2 snRNPs, Lea1 contacts Prp39 through a small interface (Fig. 5B, left panel, and fig. S7B). An RNA duplex from U2 snRNA binds the positively charged surface of Prp42 (Fig. 5B, right panel). At the interface between U2 snRNP and the tri-snRNP, Hsh155 is positioned close to the LSm ring, and U2 snRNA connects U2 snRNP to the tri-snRNP (Fig. 5C). In addition, Hsh49 and Rse1 of the SF3b complex are positioned close to Brr2, although direct interactions may be lacking (Fig. 5D).

Fig. 5 Structural changes in the S. cerevisiae pre-B–to–B transition.

(A) Surface representation of the yeast pre-B complex. The two interfaces between U2 snRNP and the tri-snRNP are indicated by red and yellow boxes. The interface between the U1 and U2 snRNPs is indicated by a blue box. (B) The interface between the U1 and U2 snRNPs of the pre-B complex is shown in cartoon representation (left panel) and electrostatic surface potential (right panel). Lea1 directly contacts Prp39, and an RNA duplex of U2 snRNA binds the positively charged surface of Prp42. (C) A close-up view of the interface between U2 snRNP and the tri-snRNP in the pre-B complex. Hsh155 is located close to the LSm ring, and U2 snRNA links U2 snRNP to the tri-snRNP. (D) Rse1 of the SF3b complex is located close to, but may not directly interact with, Brr2 of the tri-snRNP in the pre-B complex. (E) Structural overlay of the pre-B and B complexes. Compared with its position in the pre-B complex, the entire U2 snRNP moves closer to the tri-snRNP in the B complex. The B complex is color-coded, and the pre-B complex is shown in gray. (F) A close-up view of the interface between Hsh155 and the LSm ring in the B complex. The contact surface area between Hsh155 and the LSm ring is considerably larger than in the pre-B complex. (G) A close-up view of the interface between the SF3b complex and the tri-snRNP. Cus1 and Rse1 directly contact the N-terminal and the C-terminal RecA2 domains, respectively, of Brr2 in the B complex. (H) The three B-specific proteins (Prp38, Snu23, and Spp381) stabilize the RNA elements in the B complex. (I) The positive charges on the surface of the B-specific proteins may help orient the pre-mRNA and U6 snRNA.

Compared with its position in the pre-B complex, the entire U2 snRNP is translocated toward the tri-snRNP in the B complex, resulting in a considerably more compact assembly (Fig. 5E). Brr2 undergoes a rotation of about 30° and a translocation of 40 to 50 Å in the pre-B–to–B transition. Relative to its place in the pre-B complex (Fig. 5C), Hsh155 moves closer to the LSm ring in the B complex (Fig. 5F). Cus1 and Rse1 directly interact with the N- and C-terminal RecA2 domains of Brr2, respectively (Fig. 5G). The B-specific proteins Prp38, Snu23, and Spp381 are recruited into the B complex and interact with each other (Fig. 5H). Prp38 and Snu23 are positioned close to the 5′-exon–5′SS sequences and U6 snRNA, and their positive electrostatic surface potential may help orient the 5′-end sequences of pre-mRNA (Fig. 5I).

Discussion

Structural elucidation of the pre-B complex fills an important void in the mechanistic understanding of pre-mRNA splicing by the spliceosome. The local resolutions in the core of U1 snRNP and the tri-snRNP reach 3.0 Å (fig. S5B), which allows assignment of atomic features. The pre-B complex is assembled from the A complex and the tri-snRNP. Because U1 and U2 snRNPs associate with the tri-snRNP only through transient interfaces, the interactions between U1 and U2 snRNPs in the A complex are likely preserved in the pre-B complex. Therefore, we propose that the structure of the A complex may be faithfully represented in the pre-B complex (Fig. 6A).

Fig. 6 Mechanism of assembly and activation of the spliceosome in S. cerevisiae.

(A) A proposed structure of the A complex, informed by the structure of the pre-B complex. The A complex comprises the pre-mRNA and U1 and U2 snRNPs. On the basis of this model, the U1 and U2 snRNPs are predicted to be mobile relative to each other. Two views are shown. (B) A schematic diagram of the assembly and activation of the spliceosome. GTP, guanosine triphosphate.

The determination of the pre-B structure, along with other published information, reveals the structural mechanism for assembly and activation of the S. cerevisiae spliceosome (Fig. 6B). First, the A complex associates with the U4/U6.U5 tri-snRNP to form the pre-B complex in an energy-independent manner (33). Second, driven by the ATPase/helicase Prp28 (57), U1 snRNP is dissociated, and the freed 5′-exon–5′SS sequences are relocated to the vicinity of the U6 and U5 snRNAs with the help of the B-specific proteins. Third, driven by the ATPase/helicase Brr2, the B complex undergoes a major structural arrangement to become the Bact complex (3436). This step may involve two distinct phases (Fig. 6B). In the first phase, Brr2 pulls on U4 snRNA, triggering unwinding of the U4/U6 duplex, dissociation of U4 snRNP and the LSm ring, and rearrangement of U6 snRNA and the associated protein components (3436). The B-specific proteins are also dissociated. The 5′SS is recognized by the ACAGA box of U6 snRNA, and the 5′ exon is anchored to loop I of U5 snRNA. In the second phase, about 20 proteins of the NineTeen complex (NTC) and the NTC-related complex (NTR) are recruited to stabilize the active-site RNA elements (3739). An unanticipated finding from our structure is that the RES complex, which stabilizes the pre-mRNA (38), begins to function in the pre-B complex.

Although Prp28 might be positioned in one of two candidate locations (Fig. 1A), it remains to be structurally identified in the S. cerevisiae pre-B complex. Nonetheless, the structure of the pre-B complex, together with other published information (18, 19, 40), allows us to track the movement of pre-mRNA during spliceosomal assembly and activation (fig. S10). For example, the 5′SS is translocated by about 90 Å in the pre-B–to–B transition and 40 Å in the B–to–Bact transition to form a duplex with U6 snRNA. After the structure determination of the pre-B complex presented here, the B* complex remains the only assembled spliceosome yet to be structurally characterized.

Supplementary Materials

www.sciencemag.org/content/360/6396/1423/suppl/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References (4259)

References and Notes

Acknowledgments: We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for providing facility support. The computation was completed on the Explorer 100 cluster system of the Tsinghua National Laboratory for Information Science and Technology. Funding: This work was supported by funds from the National Natural Science Foundation of China (31621092 and 31430020) and the Ministry of Science and Technology (2016YFA0501100 to J.L.). Author contributions: R.B. and R.W. purified the yeast spliceosomes and prepared the cryo-EM samples. R.B., R.W., J.L., and C.Y. collected and processed the EM data. C.Y. generated the EM map and built the atomic model. All authors contributed to structure analysis. R.B., R.W., and C.Y. contributed to manuscript preparation. Y.S. designed and guided the project and wrote the manuscript. Competing interests: The authors declare no competing financial interests. Data and materials availability: The atomic coordinates have been deposited in the Protein Data Bank with the following accession codes: 5ZWM for the tri-snRNP and U2 snRNP of the pre-B complex, 5ZWN for U1 snRNP of the pre-B complex, and 5ZWO for the B complex. The EM maps have been deposited in the EMDB with the following accession codes: EMD-6972 for the tri-snRNP and U2 snRNP of the pre-B complex, EMD-6973 for U1 snRNP of the pre-B complex, and EMD-6974 for the B complex. Requests for materials should be addressed to Y.S.

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