Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters

Database
Language
Affiliation country
Publication year range
1.
Am J Hum Genet ; 111(9): 1914-1931, 2024 Sep 05.
Article in English | MEDLINE | ID: mdl-39079539

ABSTRACT

A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N = ∼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs in TPM2, in which siRNA-mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.


Subject(s)
Alternative Splicing , Bone Density , Genome-Wide Association Study , Protein Isoforms , Proteogenomics , Quantitative Trait Loci , Humans , Protein Isoforms/genetics , Bone Density/genetics , Alternative Splicing/genetics , Proteogenomics/methods , Osteoblasts/metabolism , Polymorphism, Single Nucleotide
2.
Article in English | MEDLINE | ID: mdl-39012054

ABSTRACT

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 predefined 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 (lrRNA-seq) 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 lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoforms─a critical first step in designing functional studies and eventually clinical assays.

SELECTION OF CITATIONS
SEARCH DETAIL