Pathogenic variants that alter protein code often disrupt splicing

Soemedi, Rachel; Cygan, Kamil J.; Rhine, Christy L.; Wang, Jing; Bulacan, Charlston; Yang, John Jeongseok; Bayrak-Toydemir, Pınar; McDonald, Jamie; Fairbrother, William G.

doi:10.1038/ng.3837

Cited by 203 publications

(248 citation statements)

References 45 publications

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“…We recently developed Massively Parallel Splicing Assay (MaPSy), which enables us to directly compare the splicing performance of thousands of mutant and wildtype substrates [40, 41]. Thermodynamically less stable RNAs (higher free-energy structures) splice more efficiently, particularly those with more open (unpaired) bases in the splice-site regions (Figure 5A,B).…”

Section: Disease Mutations That Alter Rna Secondary Structuresmentioning

confidence: 99%

The effects of structure on pre-mRNA processing and stability

Soemedi

Cygan

Rhine

et al. 2017

Methods

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Pre-mRNA molecules can form a variety of structures, and both secondary and tertiary structures have important effects on processing, function and stability of these molecules. The prediction of RNA secondary structure is a challenging problem and various algorithms that use minimum free energy, maximum expected accuracy and comparative evolutionary based methods have been developed to predict secondary structures. However, these tools are not perfect, and this remains an active area of research. The secondary structure of pre-mRNA molecules can have an enhancing or inhibitory effect on pre-mRNA splicing. An example of enhancing structure can be found in a novel class of introns in zebrafish. About 10% of zebrafish genes contain a structured intron that forms a bridging hairpin that enforces correct splice site pairing. Negative examples of splicing include local structures around splice sites that decrease splicing efficiency and potentially cause mis-splicing leading to disease. Splicing mutations are a frequent cause of hereditary disease. The transcripts of disease genes are significantly more structured around the splice sites, and point mutations that increase the local structure often cause splicing disruptions. Post-splicing, RNA secondary structure can also affect the stability of the spliced intron and regulatory RNA interference pathway intermediates, such as pre-microRNAs. Additionally, RNA secondary structure has important roles in the innate immune defense against viruses. Finally, tertiary structure can also play a large role in pre-mRNA splicing. One example is the G-quadruplex structure, which, similar to secondary structure, can either enhance or inhibit splicing through mechanisms such as creating or obscuring RNA binding protein sites.

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Section: Disease Mutations That Alter Rna Secondary Structuresmentioning

confidence: 99%

The effects of structure on pre-mRNA processing and stability

Soemedi

Cygan

Rhine

et al. 2017

Methods

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“…However, the predictive power of these models may come almost entirely from sequence conservation rather than the mechanistic understanding of splicing 18,19 . These models predict that human genetic variation, and especially rare variation, often disrupt sequence features required for proper exon recognition, but it is difficult to verify the accuracy of these predictions at large scales Several groups have developed massively parallel reporter assays of splicing 8,14,20,21 . Most of these assays look at a small set of exons and mutate them to understand which elements are important for splicing.…”

Section: Main Textmentioning

confidence: 99%

“…Importantly, these methods have allowed us to better quantify how individual ESEs and ESSs combine to contribute to exon recognition in a small number of exon contexts, and can be used to build more general predictive models for exon splicing. Recently, a survey of disease variants within a much broader set of human exons found that~10% of these variants had exon recognition defects 20 . Despite the recent progress, there are still several limitations inherent to these large-scale approaches.…”

Section: Main Textmentioning

confidence: 99%

Many rare genetic variants have unrecognized large-effect disruptions to exon recognition

Cheung

Yao

et al. 2017

Preprint

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Any individual’s genome contains ∼4-5 million genetic variants that differ from reference, and understanding how these variants give rise to trait diversity and disease susceptibility is a central goal of human genetics1. A vast majority (96-99%) of an individual’s variants are common, though at a population level the overwhelming majority of variants are rare2–5. Because of their scarcity in an individual’s genome, rare variants that play important roles in complex traits are likely to have large functional effects6,7. Mutations that cause an exon to be skipped can have severe functional consequences on gene function, and many known disease-causing mutations reduce or eliminate exon recognition8. Here we explore the extent to which rare genetic variation in humans results in near complete loss of exon recognition. We developed a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS) that allows us to measure exon inclusion in thousands of human exons and surrounding intronic sequence simultaneously. We assayed 27,733 extant variants in the Exome Aggregation Consortium (ExAC)9 within or adjacent to 2,339 human exons, and found that 3.8% (1,050) of the variants, almost all of which were extremely rare, led to large-effect defects in exon recognition. Importantly, we find that 83% of these splice-disrupting variants (SDVs) are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate that loss of exon recognition is an important and underappreciated means by which rare variants exert large functional effects, and that MFASS enables their empirical assessment for splicing defects at scale.

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“…It has been estimated that ~4% of synonymous mutations are deleterious, since they result in aberrant splicing due to disruption of exonic splicing enhancers (25). Another study showed that ~10% of exonic disease-associated alleles disrupt splicing (26). Furthermore, it was proposed that ~22% of disease alleles that were originally classified as missense mutation may in fact affect splicing (27).…”

Section: Introductionmentioning

confidence: 99%

Pre-mRNA Splicing Modulation by Antisense Oligonucleotides

Singh

Luo

Singh

2018

Methods in Molecular Biology

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Pre-mRNA splicing, a dynamic process of intron removal and exon joining, is governed by a combinatorial control exerted by overlapping cis-elements that are unique to each exon and its flanking intronic sequences. Splicing cis-elements are usually 4-to-8-nucleotide-long linear motifs that provide binding sites for specific proteins. Pre-mRNA splicing is also influenced by secondary and higher order RNA structures that affect accessibility of splicing cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate splicing in vivo. Therefore, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of splicing in pathological conditions. Here we describe an ASO-based approach to increase the production of the full-length SMN2 mRNA in spinal muscular atrophy patient cells.

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Pathogenic variants that alter protein code often disrupt splicing

Cited by 203 publications

References 45 publications

The effects of structure on pre-mRNA processing and stability

The effects of structure on pre-mRNA processing and stability

Many rare genetic variants have unrecognized large-effect disruptions to exon recognition

Pre-mRNA Splicing Modulation by Antisense Oligonucleotides

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