Splicing of mRNA precursors is essential in the regulation of gene expression. U2AF65 recognizes the poly-pyrimidine tract and helps in the recognition of the branch point. Inactivation of fission yeast U2AF65 (Prp2) blocks splicing of most, but not all, pre-mRNAs, for reasons that are not understood. Here, we have determined genome-wide the splicing efficiency of fission yeast cells as they progress into synchronous meiosis in the presence or absence of functional Prp2. Our data indicate that in addition to the splicing elements at the 3\' end of any intron, the nucleotides immediately upstream the intron will determine whether Prp2 is required or dispensable for splicing. By changing those nucleotides in any given intron, we regulate its Prp2 dependency. Our results suggest a model in which Prp2 is required for the coordinated recognition of both intronic ends, placing Prp2 as a key regulatory element in the determination of the exon-intron boundaries.

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations.