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.     

RNA processing.

Significant progress has been made over the last year in our understanding of the roles that RNA-binding proteins play in pre-mRNA splicing, the components of the spliceosome and how these components relate to the mechanism of splicing. Of particular importance has been the sequence analysis of the first mammalian splicing factors and structural determination of an RNA-binding domain.     

Capturing splicing complexes to study structure and mechanism

At its most basic level, pre-mRNA splicing can be described as two coordinated nuclease reactions that cleave an intron at either end and result in ligation of the flanking exons. The fact that these reactions are catalyzed by a approximately 3-MDa behemoth of protein and RNA (the spliceosome) challenges most biochemical and structural approaches currently used to characterize lesser-sized enzymes. In addition to this molecular complexity, the highly dynamic nature of splicing complexes provides additional hurdles for mechanistic studies or three-dimensional structure determination. Thus, the methods used to study the spliceosome often probe individual properties of the machine, but no complete, high-resolution picture of splicing catalysis has yet emerged. To facilitate biochemical and structural studies of native splicing complexes, we recently described purification of the catalytic form of the spliceosome (known as C complex). This native complex is suitable for electron microscopic structure determination by single-particle methods. In this paper, we describe the purification in detail and discuss additional methods for trapping and analyzing other splicing complexes.     

Inhibition of mammalian spliceosome assembly and pre-mRNA splicing by peptide inhibitors of protein kinases.

Four peptides are shown to block mammalian spliceosome assembly and pre-mRNA splicing in vitro. Previously, these peptides have been shown to inhibit Ca2+-dependent calmodulin kinase II (CaMK II) via distinct mechanisms. One is a competitive inhibitor of the kinase, two interfere with autophosphorylation events, and one competes for binding to calmodulin, a CaMK II-activating protein. However, because EGTA does not inhibit splicing, the involvement of CaMK II itself in splicing is unlikely; rather, a protein similar to CaMK II may be involved in spliceosome assembly and splicing. Two of the inhibitory peptides, the calmodulin binding domain (CBD) and glycogen synthase (GS) fragment, block assembly of spliceosomal complex C. These peptides inhibited splicing if they were added to reactions any time within the first 10 min of splicing assays. No inhibition of spliceosome assembly or splicing occurred in the presence of randomized versions of the CBD or GS peptide. Additionally, the GS peptide inhibited splicing when added to assays at later time points, despite the fact that spliceosomal complex C had formed. Cumulatively, these analyses suggest that the peptides inhibit at least two distinct events in the spliceosomal cycle. The first event occurs early during in vitro splicing. For this event, prolonged incubations of splicing reactions do not result in a recovery of splicing activity. The second event occurs later and represents a slowing of an essential step, because splicing activity can be recovered in prolonged incubations. Peptides known to inhibit protein kinase A and protein kinase C had no effect on pre-mRNA splicing, underscoring the specificity of the observed inhibitory effects.     

snRNAs as the catalysts of pre-mRNA splicing

The spliceosome, the gigantic molecular machine that performs pre-mRNA splicing in eukaryotes, contains over 200 different proteins and five RNA molecules. The central role played by the spliceosomal RNAs in splicing has led to the hypothesis that, like the ribosome, the spliceosome is an RNA-centric enzyme and a relic from the RNA world. Recent structural studies have provided the first glimpses of the structural features of spliceosomal RNAs, and mutational analyses in vivo and in vitro have uncovered new functional roles for a catalytically essential domain. An emerging model for the active site of group II introns, a closely related class of natural ribozymes, is likely to provide a wealth of insights on structure and function of the active site of the spliceosome.     

A novel splicing factor, Yju2, is associated with NTC and acts after Prp2 in promoting the first catalytic reaction of pre-mRNA splicing

The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.     

The spliceosome: caught in a web of shifting interactions

Splicing is a crucial, ubiquitous and highly complex step in eukaryotic gene expression. The daunting complexity of the splicing reaction, although fascinating, has severely limited our understanding of its mechanistic details. Recent advances have begun to provide exciting new insights into the dynamic interactions that govern the function of the spliceosome, the multi-megadalton complex that performs splicing. An emerging paradigm is the presence of a succession of distinct conformational states, which are stabilized by an intricate network of interactions. Recent data suggest that even subtle changes in the composition of the interaction network can result in interconversion of the different conformational states, providing opportunities for regulation and proofreading of spliceosome function. Significant progress in proteomics has elucidated the protein composition of the spliceosome at different stages of assembly. Also, the increased sophistication and resolution of cryo-electron microscopy techniques, combined with high-resolution structural studies on a smaller scale, promise to create detailed images of the global structure of the spliceosome and its main components, which in turn will provide a plethora of mechanistic insights. Overall, the past two years have seen a convergence of data from different lines of research into what promises to become a holistic picture of spliceosome function.     

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.     

A novel function for human factor C1 (HCF-1), a host protein required for herpes simplex virus infection,in pre-mRNA splicing

Human factor C1 (HCF-1) is needed for the expression of herpes simplex virus 1 (HSV-1) immediate-early genes in infected mammalian cells. Here, we provide evidence that HCF-1 is required for spliceosome assembly and splicing in mammalian nuclear extracts. HCF-1 interacts with complexes containing splicing snRNPs in uninfected mammalian cells and is a stable component of the spliceosome complex. We show that a missense mutation in HCF-1 in the BHK21 hamster cell line tsBN67, at the non-permissive temperature, inhibits the protein's interaction with U1 and U5 splicing snRNPs, causes inefficient spliceosome assembly and inhibits splicing. Transient expression of wild-type HCF-1 in tsBN67 cells restores splicing at the non-permissive temperature. The inhibition of splicing in tsBN67 cells correlates with the temperature-sensitive cell cycle arrest phenotype, suggesting that HCF-1-dependent splicing events may be required for cell cycle progression.     

New insights into the spliceosome by single molecule fluorescence microscopy

Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ～100 proteins minimally required for activity. Because of the spliceosome's size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing.     

RNA splicing: What has phosphorylation got to do with it?

Many pre-mRNA splicing factors are phosphorylated in vivo, but the role of this modification has been unclear. Recent observations suggest that phosphorylation modulates protein-protein interactions within the spliceosome, thereby contributing to dynamic structural reorganization of the spliceosome during splicing.     

Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing

The spliceosome is thought to undergo a conformational change between the two catalytic steps of precursor messenger RNA splicing, although the specific events in this transition are poorly understood. We previously proposed a two-state model of splicing in which the conformations required for the first and second steps are in competition. Here, we identify and characterize a class of prp8 mutants that suppress first-step splicing defects and oppose the action of the previously described prp8 suppressors of second-step defects; these opposing effects parallel those of ribosomal 'ram' and 'restrictive' mutants, which alter fidelity of transfer RNA decoding. On the basis of genetic interactions, we propose that prp8-mediated substrate repositioning during the transition occurs between catalytic-center opening and closure mediated by the U6 small nuclear RNA and the DExH/D ATPase gene prp16. Modulation of these events alters splice-site selection and splicing fidelity.     

Functional roles of DExD/H-box RNA helicases in Pre-mRNA splicing

Splicing of precursor mRNA takes place via two consecutive steps of transesterification catalyzed by a large ribonucleoprotein complex called the spliceosome. The spliceosome is assembled through ordered binding to the pre-mRNA of five small nuclear RNAs and numerous protein factors, and is disassembled after completion of the reaction to recycle all components. Throughout the splicing cycle, the spliceosome changes its structure, rearranging RNA-RNA, RNA-protein and protein-protein interactions, for positioning and repositioning of splice sites. DExD/H-box RNA helicases play important roles in mediating structural changes of the spliceosome by unwinding of RNA duplexes or disrupting RNA-protein interactions. DExD/H-box proteins are also implicated in the fidelity control of the splicing process at various steps. This review summarizes the functional roles of DExD/H-box proteins in pre-mRNA splicing according to studies conducted mostly in yeast and will discuss the concept of the complicated splicing reaction based on recent findings.     

Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages

The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1''s role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.