Fenix, A. | Search bioRxiv

bioRxiv2024

Nascent transcript O-MAP reveals the molecular architecture of a single-locus subnuclear compartment built by RBM20 and the TTN RNA

Eukaryotic nuclei adopt a highly compartmentalized architecture that influences nearly all genomic processes. Understanding how this architecture impacts gene expression has been hindered by a lack of tools for elucidating the molecular interactions at individual genomic loci. Here, we adapt oligonucleotide-mediated proximity-interactome mapping (O-MAP) to biochemically characterize discrete, micron-scale nuclear neighborhoods. By targeting O-MAP to introns within the TTN pre-mRNA, we systematically map the chromatin loci, RNAs, and proteins within a muscle-specific RNA factory organized around the TTN locus. This reveals an unanticipated compartmental architecture that organizes cis- and trans-interacting chromosomal domains, including a hub of transcriptionally silenced chromatin. The factory also recruits dozens of unique RNA-binding and chromatin-scaffolding factors, including QKI and SAFB, along with their target transcripts. Loss of the cardiac-specific splicing factor RBM20--a master regulator of TTN splicing that is mutated in dilated cardiomyopathy--remodels nearly every facet of this architecture. This establishes O-MAP as a pioneering method for probing single-locus, microcompartment-level interactions that are opaque to conventional tools. Our findings suggest new mechanisms by which coding genes can "moonlight" in nuclear-architectural roles.

genomics↗

bioRxiv2024

IS-PRM-based peptide targeting informed by long-read sequencing for alternative proteome detection

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of pre-defined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach, called LRP-IS-PRM, is a new modality for generating protein-level evidence of alternative isoforms - a critical first step in designing functional studies and eventually clinical assays.

molecular biology↗