Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins

Using Pre-mRNA Secondary Structure to Predict Splicing

Recently, scientists have tried to explain the mechanisms of post-transcriptional splicing. Developments have been made in mRNA secondary structure prediction programs that use thermodynamics to construct possible structural models of mRNA sequences (Seetin and Mathews 2012). Although splicing is a complex process that is affected by multiple interactive variables, attempts have been made to use pre-mRNA secondary structure to explain how splicing decisions are made. Here we show that there does exist some relationship between structure and splicing. Our analysis reveals that in certain areas of an optimized window of Wilms’ Tumor gene exon 5 with flanks, a significant correlation exists between regions of structural pairedness and splicing. Our results specifically point to a strong stem in the exon studied that retains its double-strandedness in the presence of mutations and is associated with high splicing. These findings advance our understanding of the influence of secondary structure on mRNA-protein interactions and encourage us to delve deeper into the complexities of splice site recognition

A Dissipative Supramolecular Glue for Temporal Control of Amplified Enzyme Activity and Biocatalytic Cascades

Regulation of enzyme activity is key to the adaptation of cellular processes such as signal transduction and metabolism in response to varying external conditions. Synthetic molecular glues have provided effective systems for enzyme inhibition and regulation of protein-protein interactions. So far, all the molecular glue systems based on covalent interactions operated in equilibrium conditions. To emulate dynamic far-from-equilibrium biological processes, we introduce herein a transient supramolecular glue with controllable lifetime. The transient system uses multivalent supramolecular interactions between guanidium group-bearing surfactants and adenosine triphosphates (ATP), resulting in bilayer vesicle structures. Unlike the conventional fuels for non-equilibrium assemblies, ATP here plays the dual role of providing a structural component for the assembly as well as presenting active functional groups to “glue” enzymes on the surface. While gluing of the enzymes on the vesicles achieves augmented catalysis, oscillation of ATP concentration allows temporal control of the catalytic activities. We further demonstrate temporal activation and control of biocatalytic cascade networks on the vesicles, which represents an essential cellular component. Altogether, the temporal activation of biocatalytic cascades on the dissipative vesicular glue presents an adaptable and dynamic system emulating heterogeneous cellular processes, opening up avenues for effective protocell construction and therapeutic interventions