Prp8 impacts cryptic but not alternative splicing frequency.

Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5' splice site, 3' splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or "cryptic" splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome's catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.

Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.

Prp8 is an essential protein that regulates spliceosome assembly and conformation during pre-mRNA splicing. Recent cryo-EM structures of the spliceosome model Prp8 as a scaffold for the spliceosome's catalytic U snRNA components. Using a new amino acid probing strategy, we identified a dynamic region in human Prp8 that is positioned to stabilize the pre-mRNA in the spliceosome active site through interactions with U5 snRNA. Mutagenesis of the identified Prp8 residues in yeast indicates a role in 5' splice site recognition. Genetic interactions with spliceosome proteins Isy1, which buttresses the intron branch point, and Snu114, a regulatory GTPase that directly contacts Prp8, further corroborate a role for the same Prp8 residues in substrate positioning and activation. Together the data suggest that adjustments in interactions between Prp8 and U5 snRNA help establish proper positioning of the pre-mRNA into the active site to enhance 5' splice site fidelity.

Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

Genetics and biochemistry remain essential in the structural era of the spliceosome.

The spliceosome is not a single macromolecular machine. Rather it is a collection of dynamic heterogeneous subcomplexes that rapidly interconvert throughout the course of a typical splicing cycle. Because of this, for many years the only high resolution structures of the spliceosome available were of smaller, isolated protein or RNA components. Consequently much of our current understanding of the spliceosome derives from biochemical and genetic techniques. Now with the publication of multiple, high resolution structures of the spliceosome, some question the relevance of traditional biochemical and genetic techniques to the splicing field. We argue such techniques are not only relevant, but vital for an in depth mechanistic understanding of pre-mRNA splicing.

Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing.

Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiaes how that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the "hinge" that links the Prp8 Jab1-MPN regulatory "tail" to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis.

Protein-guided RNA dynamics during early ribosome assembly.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly.

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5' domain of the 16S rRNA. In vitro, transformation of initial S4-rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4-rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein-RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4-RNA interactions guide rRNA folding and impact late steps of 30S assembly.

Assembly of the five-way junction in the ribosomal small subunit using hybrid MD-Go simulations.

Assembly of the bacterial ribosomal small subunit (SSU) begins with the folding of the five-way junction upon interaction with the primary binding protein S4. This complex contains the largest contiguous molecular signature, which is a conserved feature in all bacterial 16S rRNAs. In a previous study, we used all-atom molecular dynamics simulations to demonstrate that the co-evolving signature in the N-terminus of S4 is intrinsically disordered and capable of accelerating the binding process through a fly casting mechanism. In this paper, comparisons between the all-atom MD simulations and FRET experiments identify multiple metastable conformations of the naked five-way junction without the presence of S4. Furthermore, we capture the simultaneous folding and binding of the five-way junction and r-protein S4 by use of a structure-based Go potential implemented within the framework of the all-atom molecular dynamics CHARMM force field. Different folding pathways are observed for the refolding of the five-way junction upon partial binding of S4. Our simulations illustrate the complex nature of RNA folding in the presence of a protein binding partner and provide insight into the role of population shift and the induced fit mechanisms in the protein:RNA folding and binding process.

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4.

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5' domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.