Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.Published versio

Linking Proteomic and Transcriptional Data through the Interactome and Epigenome Reveals a Map of Oncogene-induced Signaling

Cellular signal transduction generally involves cascades of post-translational protein modifications that rapidly catalyze changes in protein-DNA interactions and gene expression. High-throughput measurements are improving our ability to study each of these stages individually, but do not capture the connections between them. Here we present an approach for building a network of physical links among these data that can be used to prioritize targets for pharmacological intervention. Our method recovers the critical missing links between proteomic and transcriptional data by relating changes in chromatin accessibility to changes in expression and then uses these links to connect proteomic and transcriptome data. We applied our approach to integrate epigenomic, phosphoproteomic and transcriptome changes induced by the variant III mutation of the epidermal growth factor receptor (EGFRvIII) in a cell line model of glioblastoma multiforme (GBM). To test the relevance of the network, we used small molecules to target highly connected nodes implicated by the network model that were not detected by the experimental data in isolation and we found that a large fraction of these agents alter cell viability. Among these are two compounds, ICG-001, targeting CREB binding protein (CREBBP), and PKF118–310, targeting β-catenin (CTNNB1), which have not been tested previously for effectiveness against GBM. At the level of transcriptional regulation, we used chromatin immunoprecipitation sequencing (ChIP-Seq) to experimentally determine the genome-wide binding locations of p300, a transcriptional co-regulator highly connected in the network. Analysis of p300 target genes suggested its role in tumorigenesis. We propose that this general method, in which experimental measurements are used as constraints for building regulatory networks from the interactome while taking into account noise and missing data, should be applicable to a wide range of high-throughput datasets.National Science Foundation (U.S.) (DB1-0821391)National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant R01-GM089903)National Institutes of Health (U.S.) (P30-ES002109

Evidence of Extensive Alternative Splicing in Post Mortem Human Brain HTT Transcription by mRNA Sequencing - Fig 1

<p>A) Read pileup of the superset reads showing coverage in exons and specific introns. Canonical HTT gene model is in blue. B) Relative contribution of reads from each disease dataset to the superset binned into exon (top) and intron (bottom) features. Each bar is the average number of covered bases per base position in the given feature divided by the number of samples in the corresponding condition. The canonical gene model lies between the bar charts. High intronic coverage is apparent in introns 9, 10, 12, 41, 49, and 58, highlighted in grey. None of the features are obviously biased toward any of the conditions. The rightward skew of counts is indicative of the poly-A selection method used in library prep.</p

Detected Alternative Splice Events.

<p>Twelve of the 25 alternative splicing (AS) events detected using the superset reads, as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141298#pone.0141298.g002" target="_blank">Fig 2</a>. All AS forms are much less abundant than the canonical splice form. Read Support column lists the number of junction reads supporting the splice junction, with the percentage of total junction reads involved in this event listed in parentheses. The remaining events are included as processed data file GSE71191_all_merged_HTT.bed.gz in the GEO accession GSE71191.</p><p>Detected Alternative Splice Events.</p

Splicing events detected in reads of both novel and previously reported splice forms.

<p>Grey areas indicate overall aligned read coverage in the region, black areas are the spliced reads that contribute to the splicing events. Blue lines indicate detected splicing events with a minimum of 10 supporting reads. The blue track across the top of all plots is the canonical HTT splice form, with red boxes indicating the position in the gene region displayed. Splicing patterns shown in B, E, and F support the spliceforms HTT-d13, HTT-41b, and HTT-d46 reported in Ruzo et al. In C, the skipped exon 28 is consistent with an isoform identified in mouse and human [Hughes JMB 2014] but, in these data, is only seen in a splice pattern where exon 27 is also skipped.</p

APOER2 splicing repertoire in Alzheimer's disease: Insights from long-read RNA sequencing.

Disrupted alternative splicing plays a determinative role in neurological diseases, either as a direct cause or as a driver in disease susceptibility. Transcriptomic profiling of aged human postmortem brain samples has uncovered hundreds of aberrant mRNA splicing events in Alzheimer's disease (AD) brains, associating dysregulated RNA splicing with disease. We previously identified a complex array of alternative splicing combinations across apolipoprotein E receptor 2 (APOER2), a transmembrane receptor that interacts with both the neuroprotective ligand Reelin and the AD-associated risk factor, APOE. Many of the human APOER2 isoforms, predominantly featuring cassette splicing events within functionally important domains, are critical for the receptor's function and ligand interaction. However, a comprehensive repertoire and the functional implications of APOER2 isoforms under both physiological and AD conditions are not fully understood. Here, we present an in-depth analysis of the splicing landscape of human APOER2 isoforms in normal and AD states. Using single-molecule, long-read sequencing, we profiled the entire APOER2 transcript from the parietal cortex and hippocampus of Braak stage IV AD brain tissues along with age-matched controls and investigated several functional properties of APOER2 isoforms. Our findings reveal diverse patterns of cassette exon skipping for APOER2 isoforms, with some showing region-specific expression and others unique to AD-affected brains. Notably, exon 15 of APOER2, which encodes the glycosylation domain, showed less inclusion in AD compared to control in the parietal cortex of females with an APOE ɛ3/ɛ3 genotype. Also, some of these APOER2 isoforms demonstrated changes in cell surface expression, APOE-mediated receptor processing, and synaptic number. These variations are likely critical in inducing synaptic alterations and may contribute to the neuronal dysfunction underlying AD pathogenesis