The variables on RNA molecules: concert or cacophony? Answers in long-read sequencing

Foord, Careen; Hsu, Justine; Jarroux, Julien; Hu, Wen Yang; Belchikov, Natan; Pollard, Shaun; He, Yi; Joglekar, Anoushka; Tilgner, Hagen U

doi:10.1038/s41592-022-01715-9

Cited by 20 publications

(16 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These approaches have proven reliable as a way to globally quantify individual splicing events, and even in a reference annotation-free manner to discover novel splicing events. But, this information is only a partial picture-a majority of human genes express isoforms with multiple, distinct splicing events that can influence each other in cis, creating dependencies of splicing choices within the same transcript 11,12 . Unfortunately, short-read RNA-seq datasets can only return a probabilistic, not definitive, knowledge of isoform expression, many times with inaccuracies 13 .…”

Section: Introductionmentioning

confidence: 99%

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

Abood

Mesner

Jefferey

et al. 2023

Preprint

View full text Add to dashboard Cite

A major fraction of loci identified by genome-wide association studies (GWASs) lead to alterations in alternative splicing, but interpretation of how such alterations impact proteins is hindered by the technical limitations of short-read RNA-seq, which cannot directly link splicing events to full-length transcript or protein isoforms. Long-read RNA-seq represents a powerful tool to define and quantify transcript isoforms, and recently, infer protein isoform existence. Here we present a novel approach that integrates information from GWAS, splicing QTL (sQTL), 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 which colocalized with BMD associations (H4PP ≥ 0.75). We generated deep coverage PacBio long-read RNA-seq data (N=~22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were novel. By casting the colocalized sQTLs directly onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Using these data, we created one of the first proteome-scale resources defining full-length isoforms impacted by colocalized sQTLs. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense mediated decay (NMD) and 190 that potentially resulted in the expression of new protein isoforms. Finally, we identified colocalizing sQTLs in TPM2 for splice junctions between two mutually exclusive exons, and two different transcript termination sites, making it impossible to interpret without long-read RNA-seq data. siRNA mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization. We expect our approach to be widely generalizable across diverse clinical traits and accelerate system-scale analyses of protein isoform activities modulated by GWAS loci.

show abstract

Section: Introductionmentioning

confidence: 99%

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

Abood

Mesner

Jefferey

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Alternative splicing is a fundamental form of tissue-specific gene regulation which is present in >90% of multi-exon genes in humans ( 9 ). RNA transcript diversity is a result of the combinatorial effects of alternative transcription start sites (TSS), regulated exon inclusion/exclusion or intron retention, and distinct transcript termination sites due to alternative polyadenylation (APA) ( 10 ). In individual cases, synaptic genes such as Nrnx1 have been shown to have thousands of unique isoforms ( 11 ).…”

Section: Main Textmentioning

confidence: 99%

“…The recent development of third-generation long-read sequencing technologies now enables highly accurate, in-depth characterization of the full-length alternatively spliced transcriptome at scale ( 10, 31 ). Coupled with single-cell barcoding technologies and unique molecular indexing, this can be used to catalog the isoform-centric transcriptome with single cell resolution ( 20, 32 ).…”

Section: Main Textmentioning

confidence: 99%

Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms

Patowary

Zhang

Jops

et al. 2023

Preprint

View full text Add to dashboard Cite

Human brain development is under tight molecular genetic control and the recent advent of single-cell genomics has revolutionized our ability to elucidate the diverse underlying cell-types and states. Although RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, previous work has not systematically investigated the role of cell-type-specific splicing or transcript-isoform diversity during human brain development. Here, we leverage single molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution. We identify 214,516 unique isoforms, corresponding to 22,391 genes. Remarkably, we find that 72.6% of these are novel and together with >7,000 novel-spliced exons expands the proteome by 92,422 proteoforms. We uncover myriad novel isoform switches during cortical neurogenesis, implicating previously-uncharacterized RNA-binding protein-mediated and other regulatory mechanisms in cellular identity and disease. Early-stage excitatory neurons exhibit the greatest isoform diversity and isoform-based single-cell clustering identifies previously uncharacterized cell states. Leveraging this resource, we re-prioritize thousands of rare de novo risk variants associated with neurodevelopmental disorders (NDDs) and reveal that risk genes are strongly associated with the number of unique isoforms observed per gene. Altogether, this work uncovers a substantial contribution of transcript-isoform diversity in cellular identity in the developing neocortex, elucidates novel genetic risk mechanisms for neurodevelopmental and neuropsychiatric disorders, and provides a comprehensive isoform-centric gene annotation for the developing human brain.

show abstract

“…Long-read sequencing is an emerging technology that has made important contributions to RNA biology since its inception [16][17][18][19][20] . Long-read single-cell and single-nuclei sequencing in fresh 21,22 and frozen 23 tissue allows the study of alternative splicing at the cell-type level in the brain and other complex tissues.…”

Section: Introductionmentioning

confidence: 99%

Predicting cell-type-specific exon inclusion in the human brain reveals more complex splicing mechanisms in neurons than glia

Michielsen,

Hsu,

Joglekar

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Alternative splicing contributes to molecular diversity across brain cell types. RNA-binding proteins (RBPs) regulate splicing, but the genome-wide mechanisms remain poorly understood. Here, we used RBP binding sites and/or the genomic sequence to predict exon inclusion in neurons and glia as measured by long-read single-cell data in human hippocampus and frontal cortex. We found that alternative splicing is harder to predict in neurons compared to glia in both brain regions. Comparing neurons and glia, the position of RBP binding sites in alternatively spliced exons in neurons differ more from non-variable exons indicating distinct splicing mechanisms. Model interpretation pinpointed RBPs, including QKI, potentially regulating alternative splicing between neurons and glia. Finally, using our models, we accurately predict and prioritize the effect of splicing QTLs. Taken together, our models provide new insights into the mechanisms regulating cell-type-specific alternative splicing and can accurately predict the effect of genetic variants on splicing.

show abstract

The variables on RNA molecules: concert or cacophony? Answers in long-read sequencing

Cited by 20 publications

References 46 publications

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms

Predicting cell-type-specific exon inclusion in the human brain reveals more complex splicing mechanisms in neurons than glia

Contact Info

Product

Resources

About