Ruixue Wan

Structure of a yeast spliceosome at 3.6-angstrom resolution

Structure and function of the spliceosome When RNA is transcribed from DNA in the eukaryotic cell nucleus, the initial transcript includes noncoding introns that must be spliced out. This splicing is done by a complex macromolecular machine, the spliceosome, which comprises five small nuclear RNAs and more than 100 associated proteins. Now, two papers reveal insights into the structure and function of the yeast spliceosome. Yan et al. describe a high-resolution structure determined by electron microscopy of a spliceosome complex comprising four RNAs and 37 proteins. Hang et al. focus on the catalytic site and show how protein components anchor the transcribed RNA and allow sufficient flexibility to deliver RNA components involved in catalyzing the splicing reaction. Science, this issue pp. 1182 and 1191 A high-resolution structure determined by electron microscopy provides insight into how the spliceosome functions. Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat. The atomic model includes 10,574 amino acids from 37 proteins and four RNA molecules, with a combined molecular mass of approximately 1.3 megadaltons. Spp42 (Prp8 in Saccharomyces cerevisiae), the key protein component of the U5 snRNP, forms a central scaffold and anchors the catalytic center. Both the morphology and the placement of protein components appear to have evolved to facilitate the dynamic process of pre-mRNA splicing. Our near-atomic-resolution structure of a central spliceosome provides a molecular framework for mechanistic understanding of pre-mRNA splicing.

Structural basis of pre-mRNA splicing

Structure and function of the spliceosome When RNA is transcribed from DNA in the eukaryotic cell nucleus, the initial transcript includes noncoding introns that must be spliced out. This splicing is done by a complex macromolecular machine, the spliceosome, which comprises five small nuclear RNAs and more than 100 associated proteins. Now, two papers reveal insights into the structure and function of the yeast spliceosome. Yan et al. describe a high-resolution structure determined by electron microscopy of a spliceosome complex comprising four RNAs and 37 proteins. Hang et al. focus on the catalytic site and show how protein components anchor the transcribed RNA and allow sufficient flexibility to deliver RNA components involved in catalyzing the splicing reaction. Science, this issue pp. 1182 and 1191 A high-resolution structure determined by electron microscopy provides insight into how the spliceosome functions. Splicing of precursor messenger RNA is performed by the spliceosome. In the cryogenic electron microscopy structure of the yeast spliceosome, U5 small nuclear ribonucleoprotein acts as a central scaffold onto which U6 and U2 small nuclear RNAs (snRNAs) are intertwined to form a catalytic center next to Loop I of U5 snRNA. Magnesium ions are coordinated by conserved nucleotides in U6 snRNA. The intron lariat is held in place through base-pairing interactions with both U2 and U6 snRNAs, leaving the variable-length middle portion on the solvent-accessible surface of the catalytic center. The protein components of the spliceosome anchor both 5′ and 3′ ends of the U2 and U6 snRNAs away from the active site, direct the RNA sequences, and allow sufficient flexibility between the ends and the catalytic center. Thus, the spliceosome is in essence a protein-directed ribozyme, with the protein components essential for the delivery of critical RNA molecules into close proximity of one another at the right time for the splicing reaction.

Structure of a yeast activated spliceosome at 3.5 Å resolution

How spliceosomes make the first cut In eukaryotes, transcribed precursor mRNA includes noncoding sequences that must be spliced out. This is done by the spliceosome, a dynamic complex in which five small nuclear RNAs and several proteins go through a series of ordered interactions and conformational rearrangements to achieve splicing. Two protein structures provide a look at the first catalytic step in the pathway. Yan et al. report the structure of the activated spliceosome (the Bact complex) at 3.5 Å resolution, revealing how latency is maintained even though the complex is mostly primed for catalysis. Wan et al. report the structure of the catalytic step 1 spliceosome (the C complex) at 3.4 Å resolution; this complex forms after the first step of the splicing reaction. Science, this issue pp. 904 and 895 Two structures give insight into the molecular basis of splicing. Pre–messenger RNA (pre-mRNA) splicing is carried out by the spliceosome, which undergoes an intricate assembly and activation process. Here, we report an atomic structure of an activated spliceosome (known as the Bact complex) from Saccharomyces cerevisiae, determined by cryo–electron microscopy at an average resolution of 3.52 angstroms. The final refined model contains U2 and U5 small nuclear ribonucleoprotein particles (snRNPs), U6 small nuclear RNA (snRNA), nineteen complex (NTC), NTC-related (NTR) protein, and a 71-nucleotide pre-mRNA molecule, which amount to 13,505 amino acids from 38 proteins and a combined molecular mass of about 1.6 megadaltons. The 5ʹ exon is anchored by loop I of U5 snRNA, whereas the 5ʹ splice site (5ʹSS) and the branch-point sequence (BPS) of the intron are specifically recognized by U6 and U2 snRNA, respectively. Except for coordination of the catalytic metal ions, the RNA elements at the catalytic cavity of Prp8 are mostly primed for catalysis. The catalytic latency is maintained by the SF3b complex, which encircles the BPS, and the splicing factors Cwc24 and Prp11, which shield the 5ʹ exon–5ʹSS junction. This structure, together with those determined earlier, outlines a molecular framework for the pre-mRNA splicing reaction.

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

How spliceosomes make the first cut In eukaryotes, transcribed precursor mRNA includes noncoding sequences that must be spliced out. This is done by the spliceosome, a dynamic complex in which five small nuclear RNAs and several proteins go through a series of ordered interactions and conformational rearrangements to achieve splicing. Two protein structures provide a look at the first catalytic step in the pathway. Yan et al. report the structure of the activated spliceosome (the Bact complex) at 3.5 Å resolution, revealing how latency is maintained even though the complex is mostly primed for catalysis. Wan et al. report the structure of the catalytic step 1 spliceosome (the C complex) at 3.4 Å resolution; this complex forms after the first step of the splicing reaction. Science, this issue pp. 904 and 895 Two structures give insight into the molecular basis of splicing. Each cycle of pre–messenger RNA splicing, carried out by the spliceosome, comprises two sequential transesterification reactions, which result in the removal of an intron and the joining of two exons. Here we report an atomic structure of a catalytic step I spliceosome (known as the C complex) from Saccharomyces cerevisiae, as determined by cryo–electron microscopy at an average resolution of 3.4 angstroms. In the structure, the 2′-OH of the invariant adenine nucleotide in the branch point sequence (BPS) is covalently joined to the phosphate at the 5′ end of the 5′ splice site (5′SS), forming an intron lariat. The freed 5′ exon remains anchored to loop I of U5 small nuclear RNA (snRNA), and the 5′SS and BPS of the intron form duplexes with conserved U6 and U2 snRNA sequences, respectively. Specific placement of these RNA elements at the catalytic cavity of Prp8 is stabilized by 15 protein components, including Snu114 and the splicing factors Cwc21, Cwc22, Cwc25, and Yju2. These features, representing the conformation of the spliceosome after the first-step reaction, predict structural changes that are needed for the execution of the second-step transesterification reaction.

The 3.8 Å structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis

Structure of a key spliceosomal complex In eukaryotes, when DNA is transcribed into RNA, the primary transcript has protein-coding sequences interrupted by noncoding sequences called introns. Introns are removed by a complex molecular machine, the spliceosome. Wan et al. determined the structure of a key subcomplex, the tri-SNP, that comprises three small nuclear RNAs and more than 30 proteins. The structure, determined by electron microscopy at 3.8 Å resolution, unexpectedly shows a primary RNA transcript bound in the tri-SNP. Further analysis revealed how the spliceosome assembles to achieve its complex functions. Science, this issue p. 466 A key spliceosomal subcomplex reveals how the spliceosome assembles for activity. Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which comprises the U5 small nuclear ribonucleoprotein (snRNP), the U4 and U6 small nuclear RNA (snRNA) duplex, and a number of protein factors. Here we report the three-dimensional structure of a Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at an overall resolution of 3.8 angstroms by single-particle electron cryomicroscopy. The local resolution for the core regions of the tri-snRNP reaches 3.0 to 3.5 angstroms, allowing construction of a refined atomic model. Our structure contains U5 snRNA, the extensively base-paired U4/U6 snRNA, and 30 proteins including Prp8 and Snu114, which amount to 8495 amino acids and 263 nucleotides with a combined molecular mass of ~1 megadalton. The catalytic nucleotide U80 from U6 snRNA exists in an inactive conformation, stabilized by its base-pairing interactions with U4 snRNA and protected by Prp3. Pre-messenger RNA is bound in the tri-snRNP through base-pairing interactions with U6 snRNA and loop I of U5 snRNA. This structure, together with that of the spliceosome, reveals the molecular choreography of the snRNAs in the activation process of the spliceosomal ribozyme.

Structure of a yeast step II catalytically activated spliceosome

Poised for the second step of splicing In eukaryotes, noncoding sequences in transcribed precursor mRNA are cut out by a dynamic macromolecular machine, the spliceosome. This involves two sequential reactions. The first cuts one end of the noncoding intron and loops it back on itself to form an intron lariat, and the next excises the intron and ligates the coding mRNA. Insights into the first step of splicing have come from the structures of two intermediates: the Bact complex, which is primed for catalysis, and the C complex, which is formed after the first splicing reaction. Yan et al. now report a high-resolution structure of the step II catalytically activated spliceosome (the C* complex). This structure shows conformational changes that position catalytic motifs to accomplish the second splicing reaction. Science, this issue p. 149 A spliceosome intermediate complex is poised to complete the second step of splicing. Each cycle of precursor messenger RNA (pre-mRNA) splicing comprises two sequential reactions, first freeing the 5′ exon and generating an intron lariat–3′ exon and then ligating the two exons and releasing the intron lariat. The second reaction is executed by the step II catalytically activated spliceosome (known as the C* complex). Here, we present the cryo–electron microscopy structure of a C* complex from Saccharomyces cerevisiae at an average resolution of 4.0 angstroms. Compared with the preceding spliceosomal complex (C complex), the lariat junction has been translocated by 15 to 20 angstroms to vacate space for the incoming 3′-exon sequences. The step I splicing factors Cwc25 and Yju2 have been dissociated from the active site. Two catalytic motifs from Prp8 (the 1585 loop and the β finger of the ribonuclease H–like domain), along with the step II splicing factors Prp17 and Prp18 and other surrounding proteins, are poised to assist the second transesterification. These structural features, together with those reported for other spliceosomal complexes, yield a near-complete mechanistic picture on the splicing cycle.

Crystal structures of the Lsm complex bound to the 3' end sequence of U6 small nuclear RNA.

Splicing of precursor messenger RNA (pre-mRNA) in eukaryotic cells is carried out by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs) and a number of accessory factors and enzymes. Each snRNP contains a ring-shaped subcomplex of seven proteins and a specific RNA molecule. The U6 snRNP contains a unique heptameric Lsm protein complex, which specifically recognizes the U6 small nuclear RNA at its 3′ end. Here we report the crystal structures of the heptameric Lsm complex, both by itself and in complex with a 3′ fragment of U6 snRNA, at 2.8 Å resolution. Each of the seven Lsm proteins interacts with two neighbouring Lsm components to form a doughnut-shaped assembly, with the order Lsm3–2–8–4–7–5–6. The four uridine nucleotides at the 3′ end of U6 snRNA are modularly recognized by Lsm3, Lsm2, Lsm8 and Lsm4, with the uracil base specificity conferred by a highly conserved asparagine residue. The uracil base at the extreme 3′ end is sandwiched by His 36 and Arg 69 from Lsm3, through π–π and cation–π interactions, respectively. The distinctive end-recognition of U6 snRNA by the Lsm complex contrasts with RNA binding by the Sm complex in the other snRNPs. The structural features and associated biochemical analyses deepen mechanistic understanding of the U6 snRNP function in pre-mRNA splicing.

Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation

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 Cryo–electron microscopy structures of the pre-B and B complexes reveal the mechanism of assembly and activation for the yeast spliceosome. 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.