We use deep sequencing to identify sources of variation in mRNA splicing in the dorsolateral prefrontal cortex (DLFPC) of 450 subjects from two aging cohorts. Hundreds of aberrant pre-mRNA splicing events are reproducibly associated with Alzheimer’s disease. We also generate a catalog of splicing quantitative trait loci (sQTL) effects: splicing of 3,006 genes is influenced by genetic variation. We report that altered splicing is the mechanism for the effects of the PICALM, CLU , and PTK2B susceptibility alleles. Further, we performed a transcriptome-wide association study and identified 21 genes with significant associations to Alzheimer’s disease, many of which are found in known loci, but 8 are in novel loci. This highlights the convergence of old and new Alzheimer’s disease genes in autophagy-lysosomal-related pathways. Overall, this study of the aging brain’s transcriptome provides evidence that dysregulation of mRNA splicing is a feature of Alzheimer’s disease and is, in some cases, genetically driven.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes 1-11 , but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far 12,13. Although single splicing events have been described for ≤200 single cells with statistical confidence 14,15 , full-length mRNA analyses for hundreds of cells have not been reported. Singlecell short-read 3′ sequencing enables the identification of cellular subtypes 16-21 , but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-typespecific combination patterns of distant splice sites 6-9,22,23. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms. Unlike sorting-based methods (Supplementary Fig. 1a), ScISOr-Seq identifies isoforms in >1,000 single cells from bulk tissue without cell sorting by combining two technologies (Fig. 1a). We used microfluidics to amplify full-length cDNA from single cells in a sample. cDNA produced from each single cell was barcoded to enable cell-of-origin identification and then split into two pools, with one pool being used for short-read Illumina 3′ sequencing to measure gene expression and the other pool being used for long-read sequencing and isoform identification. Short-read 3′ sequencing provided molecular counts for each gene and cell, which enabled clustering of cells and cell type assignment using cell-type-specific markers. Long-read sequencing with Pacific Biosciences (PacBio) 1,2,4,5 or Oxford Nanopore 3 was used to identify full-length RNA isoforms. Single-cell barcodes were also present in long reads and could be used to determine the individual

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.

Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3 0 transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3 0 untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3 0 UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA-protein interactions in conditioning isoform-specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.