Recurrent Spliceosome Mutations in Cancer: Mechanisms and Consequences of Aberrant Splice Site Selection

Splicing alterations have been widely documented in tumors where the proliferation and dissemination of cancer cells is supported by the expression of aberrant isoform variants. Splicing is catalyzed by the spliceosome, a ribonucleoprotein complex that orchestrates the complex process of intron removal and exon ligation. In recent years, recurrent hotspot mutations in the spliceosome components U1 snRNA, SF3B1, and U2AF1 have been identified across different tumor types. Such mutations in principle are highly detrimental for cells as all three spliceosome components are crucial for accurate splice site selection: the U1 snRNA is essential for 3′ splice site recognition, and SF3B1 and U2AF1 are important for 5′ splice site selection. Nonetheless, they appear to be selected to promote specific types of cancers. Here, we review the current molecular understanding of these mutations in cancer, focusing on how they influence splice site selection and impact on cancer development.

psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3′-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.

Structural basis for conformational equilibrium of the catalytic spliceosome

The catalytic spliceosome exists in equilibrium between the branching (B*/ C) and exon ligation (C*/ P) conformations. Here we present the electron cryo-microscopy reconstruction of the Saccharomyces cerevisiae C complex at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. In the Ci conformation, the exon-ligation factors Prp18 and Slu7 are already bound before ATP hydrolysis by Prp16, which destabilises the branching conformation. Biochemical assays suggest these pre-bound factors prime C complex for conversion to C* by Prp16. A complete model of the Prp19-complex (NTC) shows how the NTC pre-recruits the branching factors Yju2 and Isy1 before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain associated with the C* and P spliceosomes and promote exon ligation. Our results explain how Prp16 action modulates dynamic binding of step-specific factors to alternatively stabilise the C or C* conformation and establish equilibrium of the catalytic spliceosome.

How Is Precursor Messenger RNA Spliced by the Spliceosome?

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.

Design of a GFP reporter for splicing analysis in mammalian cells

Objective: The great majority of eukaryotic genes are formed by exons and introns. Pre-RNA transcripts are extensively processed in the nucleus, with the addition of a cap group at the 5′ end, intron removal and exon ligation (splicing) followed by addition of a poly-A tail at 3′ end. Splicing is performed by specialized macromolecular machinery named spliceosome, composed of five small ribonucleoprotein particles (snRNPs) and several proteins. The activity of this complex is highly accurate due to coordinated activity of its components. Altered splicing has already been related to the development of several diseases as amyotrophic lateral sclerosis and different types of cancer. Detailed understanding of splicing regulation in eukaryotic cells can be achieved using splicing reporter systems. Results: We designed a new splicing reporter plasmid suitable for analysis in mammalian cells. Our reporter is based on splicing of the GFP sequence. The greatest advantages of this system are the ease of visualization of the splicing outcome, by using a fluorescence microscope to confirm GFP expression from the reporter. Also, quantification of splicing efficiency using real-time PCR is possible. The use of this system allows rapid and easy detection of the splicing results in cultured cells.

Design of a GFP reporter for splicing analysis in mammalian cells

Objective: The great majority of eukaryotic genes are formed by exons and introns. Pre-RNA transcripts are extensively co-transcriptionally processed, with the addition of a cap group at the 5′ end, intron removal and exon ligation (splicing) followed by addition of a poly-A tail at 3′ end. Splicing is performed by specialized macromolecular machinery named spliceosome, composed of five small ribonucleoprotein particles (snRNPs) and several proteins. The activity of this complex is highly accurate due to coordinated activity of its components. Altered splicing have already been related to the development of several diseases as amyotrophic lateral sclerosis and different types of cancer. Detailed understanding of splicing regulation in eukaryotic cells can be achieved using splicing reporter systems. Results: We designed a new splicing reporter plasmid suitable for analysis in mammalian cells. Our reporter is based on splicing of the GFP sequence. The greatest advantages of this system are the ease of visualization of the splicing reaction, which needs only a fluorescence microscope, and the possibility of quantification of splicing efficiency using real-time PCR. The use of this system allows rapid and easy detection of the splicing reactions.

PsiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

Eight RNA helicases remodel the spliceosome to effect pre-mRNA splicing but their mechanism of action remains poorly understood. We have developed “purified spliceosome iCLIP” (psiCLIP) to define helicase-RNA contacts in specific spliceosomal states. psiCLIP reveals previously unappreciated dynamics of spliceosomal helicases. The binding profile of the helicase Prp16 is influenced by the distance between the branch-point and 3’ splice site, while Prp22 binds diffusely on the intron before exon ligation but switches to more narrow binding downstream of the exon junction after exon ligation. Notably, depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, demonstrating that psiCLIP can be used to study the relationships between helicases and auxiliary splicing factors. Thus, psiCLIP is sensitive to spliceosome dynamics and complements the insights from structural and imaging studies by providing crucial positional information on helicase-RNA contacts during spliceosomal remodeling.

Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2′-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3′-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.