Splicing of designer exons informs a biophysical model for exon definition

Arias, Mauricio; Lubkin, Ashira; Chasin, Lawrence A.

doi:10.1261/rna.048009.114

Cited by 20 publications

(29 citation statements)

References 70 publications

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“…This observation can be explained by the classical exon definition model where the shortening of the exon size below 50 nt can lead to a steric clash with the spliceosomal complexes binding to the 3 0 and 5 0 ends of exon. 42,43 Together, these result suggest that the position of the USSE element in respect to upstream 3 0 ss is dictated by the physical constraints imposed by exon definition interactions. In contrast, similar distance constraints are not apparent for canonical splicing activators (i.e.…”

Section: Discussionmentioning

confidence: 81%

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

Niemelä¹,

Verbeeren

Singha

et al. 2015

RNA Biology

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Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5 0 splice sites (5 0 ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50 nt downstream from the target 3 0 splice site (3 0 ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3 0 ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5 0 ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5 0 ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.

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Section: Discussionmentioning

confidence: 81%

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

Niemelä¹,

Verbeeren

Singha

et al. 2015

RNA Biology

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“…Notably, our conclusions concerning RON splicing are robust to the precise implementation of the exon definition mechanism: in our model, we assume that U1 and U2 snRNP independently recognize splice sites, and that cross-exon and cross-intron complexes form only later during spliceosome maturation. Alternatively, exon definition may already occur at the level of initial U1 and U2 snRNP binding, because both subunits cooperate across exons during splice site recognition (9,13). In Materials and Methods, we show that both scenarios lead to the same splice isoform probability equations, implying that our fitting results also apply for strong cross-exon cooperation of U1 and U2 snRNP binding.…”

Section: High-throughput Mutagenesis Data Supports the Exon Definitiomentioning

confidence: 69%

“…To date, only a handful of mechanistic modeling studies on alternative splicing have been published. These mainly focused on the quantification of mutation effects (9,11,17), studied the impact of co-transcriptional splicing (10,18) and analyzed cell-to-cell variability of the process (19,20). Here, we approach splicing regulation from a different angle and mechanistically describe how splice site recognition by the spliceosome shapes the splicing outcome.…”

Section: Discussionmentioning

confidence: 99%

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Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

Enculescu¹,

Braun²,

Setty

et al. 2019

Preprint

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Alternative splicing is a key step in eukaryotic gene expression that allows the production of multiple protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. Using mechanistic mathematical modeling, we show that alternative splicing control is facilitated if spliceosomes recognize exons as functional units ('exon definition'). We find that exon definition is crucial to prevent the accumulation of partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-mRNA. These predictions of our model are qualitatively and quantitatively supported by high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON (MST1R). Our analysis suggests that exon definition has evolved as the dominant splice-regulatory mechanism in higher organisms to promote robust and reliable splicing outcomes.One Sentence Summary: Exon definition is required for precise alternative splicing control and prevents accumulation of undesired retention isoforms.

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“…http://dx.doi.org/10.1101/199927 doi: bioRxiv preprint first posted online Oct. 8, 2017; is usually a rarer event in higher eukaryotes that contain longer introns 32 . We chose two longer intronic backbones (~300-600 bp) shown previously to not suffer from such intronic retention ( C. griseus DHFR and human SMN1 intron backbones), and found that the longer intron lengths improved both expression and assay reproducibility 33,34 . Exon inclusion metrics obtained from both of these intron contexts were highly reproducible between biological replicates (Fig.…”

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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Splicing of designer exons informs a biophysical model for exon definition

Cited by 20 publications

References 70 publications

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

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

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