Three-dimensional structure of C complex spliceosomes by electron microscopy

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 A. The structure reveals a particle of dimensions 27 x 22 x 24 nm with a relatively open arrangement of three primary domains.     

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.     

Spliceosome structure: Piece by piece

Processing of pre-mRNAs by RNA splicing is an essential step in the maturation of protein coding RNAs in eukaryotes. Structural studies of the cellular splicing machinery, the spliceosome, are a major challenge in structural biology due to the size and complexity of the splicing ensemble. Specifically, the structural details of splice site recognition and the architecture of the spliceosome active site are poorly understood. X-ray and NMR techniques have been successfully used to address these questions defining the structure of individual domains, isolated splicing proteins, spliceosomal RNA fragments and recently the U1 snRNP multiprotein·RNA complex. These results combined with extant biochemical and genetic data have yielded important insights as well as posing fresh questions with respect to the regulation and mechanism of this critical gene regulatory process.     

Structural studies of the spliceosome: Bridging the gaps

The spliceosome is a multi-megadalton RNA–protein complex responsible for the removal of non-coding introns from pre-mRNAs. Due to its complexity and dynamic nature, it has proven to be a very challenging target for structural studies. Developments in single particle cryo-EM have overcome these previous limitations and paved the way towards a structural characterisation of the splicing machinery. Despite tremendous progress, many aspects of spliceosome structure and function remain elusive. In particular, the events leading to the definition of exon–intron boundaries, alternative and non-canonical splicing events, and cross-talk with other cellular machineries. Efforts are being made to address these knowledge gaps and further our mechanistic understanding of the spliceosome. Here, we summarise recent progress in the structural and functional analysis of the spliceosome.     

Characterization of an RNP complex that assembles on the Rous sarcoma virus negative regulator of splicing element.

We have characterized an RNP complex that assembles in nuclear extracts on the negative regulator of splicing (NRS) element from Rous sarcoma virus. While no complex was detected by native gel electrophoresis under conditions that supported spliceosome assembly, gel filtration revealed a specific ATP-independent complex that rapidly assembled on NRS RNA. No complexes were formed on non-specific RNA. Unlike the non-specific H complex, factors required for NRS complex assembly are limiting in nuclear extract. The NRS complex was not detected in reactions containing ATP and pre-formed complexes were dissociated in the presence of ATP. In addition, the assembly process was sensitive to high salt but NRS complexes were salt stable once formed. Assembly of the NRS complex appears functionally significant since mutated NRS RNAs that fail to inhibit splicing in vivo are defective for NRS complex assembly in nuclear extract. The probable relationship of the NRS complex to spliceosomal complexes is discussed.     

Structural model of the p14/SF3b155 · branch duplex complex

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.     

Pre-mRNA splicing: awash in a sea of proteins

What's in a spliceosome? More than we ever imagined, according to recent reports employing proteomics techniques to analyze this multi-megadalton machine. As of 1999, around 100 splicing factors were identified (Burge et al., 1999); however, that number has now nearly doubled due primarily to improved purification of spliceosomes coupled with advances in mass spectrometry analyses of complex mixtures. Gratifyingly, most of the previously identified splicing factors were found in the recent mass spec studies. Nonetheless, the number of new proteins emerging with no prior connection to splicing was surprising. Without functional validation, it would be premature to label these proteins as bona fide splicing factors. Yet many were identified multiple times in complexes purified under diverse conditions or from different organisms. Another recurring theme regards the dynamic nature of spliceosomal complexes, which may be even more intricate than previously thought.     

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry

Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.     

Arrested yeast splicing complexes indicate stepwise snRNP recruitment during in vivo spliceosome assembly

Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular machine dedicated to intron removal and exon ligation. Despite an abundance of in vitro information and a small number of in vivo studies, the pathway of yeast (Saccharomyces cerevisiae) in vivo spliceosome assembly remains uncertain. To address this situation, we combined in vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation (ChIP) analysis of other splicing snRNPs along an intron-containing gene. The data indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway: first U1, then U2, and finally the U4/U6*U5 tri-snRNP. Tandem affinity purification (TAP) using a U2 snRNP-tagged protein allowed the characterization of in vivo assembled higher-order splicing complexes. Consistent with an independent snRNP assembly pathway, we observed high levels of U1-U2 prespliceosomes under U5-depletion conditions, and we observed significant levels of a U2/U5/U6/Prp19-complex mature splicing complex under wild-type conditions. These complexes have implications for the steady-state distribution of snRNPs within nuclei and also reinforce the stepwise recruitment of U1, U2, and the tri-snRNP during in vivo spliceosome assembly.     

Functional roles of protein splicing factors

RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors. DExD/H-box RNA helicases are required to mediate structural changes in the spliceosome at various steps in the assembly pathway and have also been implicated in the fidelity control of the splicing reaction. Other proteins also play key roles in mediating the progression of the spliceosome pathway. In this review, we discuss the functional roles of the protein factors involved in the spliceosome pathway primarily from studies in the yeast system.     

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.     

Structural analyses of the pre‐mRNA splicing machinery

Pre‐mRNA splicing is a critical event in the gene expression pathway of all eukaryotes. The splicing reaction is catalyzed by the spliceosome, a huge protein‐RNA complex that contains five snRNAs and hundreds of different protein factors. Understanding the structure of this large molecular machinery is critical for understanding its function. Although the highly dynamic nature of the spliceosome, in both composition and conformation, posed daunting challenges to structural studies, there has been significant recent progress on structural analyses of the splicing machinery, using electron microscopy, crystallography, and nuclear magnetic resonance. This review discusses key recent findings in the structural analyses of the spliceosome and its components and how these findings advance our understanding of the function of the splicing machinery.     

Isolation and characterization of post-splicing lariat–intron complexes

Pre-mRNA splicing occurs in a large complex spliceosome. The steps of both spliceosome assembly and splicing reaction have been extensively analyzed, and many of the factors involved have been identified. However, the post-splicing intron turnover process, especially in vertebrates, remains to be examined. In this paper, we developed a two-tag affinity purification method for purifying lariat intron RNA–protein complexes obtained from an in vitro splicing reaction. Glycerol gradient sedimentation analyses revealed that there are at least two forms of post-splicing intron complexes, which we named the ‘Intron Large (IL)’ and the ‘Intron Small (IS)’ complexes. The IL complex contains U2, U5 and U6 snRNAs and other protein splicing factors, whereas the IS complex contains no such U snRNAs or proteins. We also showed that TFIP11, a human homolog of yeast Ntr1, is present in the IL complex and the TFIP11 mutant protein, which lacks the interaction domain with hPrp43 protein, caused accumulation of the IL complex and reduction of IS complex formation in vitro. Taken together, our results strongly suggest that TFIP11 in cooperation with hPrp43 mediates the transition from the IL complex to the IS complex, leading to efficient debranching and turnover of excised introns.