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Human Gene Expression

Global impact of unproductive splicing on human gene expression

Alternative splicing (AS) in human genes is widely viewed as a mechanism for enhancing proteomic diversity. AS can also impact gene expression levels without increasing protein diversity by producing ‘unproductive’ transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD). However, the relative importance of this regulatory mechanism remains underexplored. To better understand the impact of AS–NMD relative to other regulatory mechanisms, we analyzed population-scale genomic data across eight molecular assays, covering various stages from transcription to cytoplasmic decay. We report threefold more unproductive splicing compared with prior estimates using steady-state RNA. This unproductive splicing compounds across multi-intronic genes, resulting in 15% of transcript molecules from protein-coding genes being unproductive. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are as often associated with NMD-induced expression level differences as with differences in protein isoform usage. Our findings suggest that much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.

Main

Alternative splicing (AS) has the potential to expand the number of functional peptides encoded in messenger RNA. Large-scale transcriptomics studies have confirmed that nearly all protein-coding genes generate multiple—sometimes dozens—of distinct mRNA isoforms. This finding is often interpreted as supporting the role of AS in diversifying the proteome; yet, most alternatively spliced isoforms are lowly expressed and lack cross-species conservation. To explain these observations, multiple studies have suggested that the vast majority of isoforms are nonfunctional transcripts resulting from mis-splicing rather than regulated AS.

Mis-splicing from aberrant activation of unconserved ‘cryptic’ splice sites often introduces frameshifts in the mRNA coding sequence, resulting in premature termination codons (PTCs). Consequently, downstream exon junction complexes, which would normally be displaced by translating ribosomes, recruit nonsense-mediated decay (NMD) machinery to the mRNA for degradation. Thus, most transcripts with one or more aberrant splicing events are considered to be ‘unproductive’, as they are expected to undergo rapid NMD.

Unproductive transcripts can also result from regulated AS. For example, some splicing factors control AS of their own pre-mRNA, relying on the coupling between AS and NMD (AS–NMD) to autoregulate their expression levels. However, regulated AS–NMD has only been documented in a handful of genes. While most genes have annotated unproductive isoform structures, the extent to which these isoforms influence gene expression levels, impact phenotypes and/or are important for organismal fitness is unknown. Assessing these questions is complicated partly because the rapid decay of unproductive isoforms obscures quantitative measurements of their splicing.

An early study of unproductive splicing estimated that up to a third of transcript isoforms inferred from expressed sequence tags supported unproductive rather than productive splicing and hypothesized that AS–NMD may be a widespread regulatory mechanism. Since then, multiple studies have attempted to test this hypothesis using improved methods, for example, by measuring gene expression levels before and after knocking down core NMD factors such as UPF1 or UPF2. These studies revealed a modest impact on gene expression levels, noting that only a small fraction (<10%) of genes show appreciable change in gene expression levels upon knockdown, providing evidence against a widespread role of AS–NMD. By contrast, recent studies support partial redundancy between core NMD factors, which can obscure knockdowns of single NMD factors and underestimate the impact of AS–NMD on mRNA expression levels. Thus, the impact of AS on gene expression levels remains unclear.

Results

High-throughput measurements of AS before mRNA decay

To assess the impact of AS on steady-state gene expression levels, we must jointly consider multiple stages of gene regulation that reflect mRNA before and after the influence of cytoplasmic decay processes. To do this, we leveraged a large collection of molecular assays in lymphoblastoid cell lines (LCLs) derived primarily from 40–86 Yoruba individuals. These datasets have been used to study the impact of genetic variants on molecular phenotypes and consists of measurements tracking major steps of mRNA biogenesis including chromatin activity at enhancers (H3K4me1 and H3K27ac combining chromatin immunoprecipitation sequencing (ChIP-seq)) and promoters (H3K27ac and H3K4me3 ChIP-seq), newly transcribed polyA RNAs (4sU pulse-labeled for 30 or 60 min) and steady-state mRNA levels (RNA sequencing, RNA-seq). However, these data fail to capture spliced mRNA before potential cytoplasmic degradation, preventing us from capturing rapidly degraded mRNA transcripts.
COVID-19, mRNA vaccination, sickle cell disease, ASH RC Sickle Cell Research Network, immune responses, vaccine efficacy, vaccine safety, public health, personalized care, vaccination strategies, study, research, immune system, vaccine response, health outcomes, medical research, disease management, patient care, vaccination, epidemiology,

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