Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition

Amrane, Samir; Rebora, Karine; Zniber, Ilyass; Dupuy, Denis; Mackereth, Cameron D.

doi:10.1038/ncomms5595

Cited by 21 publications

(20 citation statements)

References 52 publications

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“…To date this is the most efficient technique to perform genetic screens in order to identify splicing factors that regulate those events and open the path to study the biochemical details of the regulatory interactions (Amrane et al, 2014, Kuwasako et al, 2014, Mackereth, 2014.…”

Section: Introductionmentioning

confidence: 99%

Quantitative RNA-seq meta analysis of alternative exon usage in C. elegans

Tourasse

Millet

Dupuy

2017

Preprint

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ABSTRACT:Almost twenty years after the completion of the C. elegans genome sequence, gene structure annotation is still an ongoing process with new evidence for gene variants still being regularly uncovered by additional in-depth transcriptome studies. While alternative splice forms can allow a single gene to encode several functional isoforms the question of how much spurious splicing is tolerated is still heavily debated.Here we gathered a compendium of 1,682 publicly available C. elegans RNA-seq datasets to increase the dynamic range of detection of RNA isoforms and obtained robust measurements of the relative abundance of each splicing event. While most of the splicing reads come from reproducibly detected splicing events, a large fraction of purported junctions are only supported by a very low number of reads. We devised an automated curation method that takes into account the expression level of each gene to discriminate robust splicing events from potential biological noise.We found that rarely used splice sites disproportionately come from highly expressed genes and are significantly less conserved in other nematode genomes than splice sites with a higher usage frequency.Our increased detection power confirmed trans-splicing for at least 84% of C. elegans protein coding genes. The genes for which trans-splicing was not observed are overwhelmingly low expression genes, suggesting that the mechanism is pervasive but not full captured by organismwide RNA-Seq.We generated annotated gene models including quantitative exon usage information for the entire C. elegans genome. This allows users to visualize at a glance the relative expression of each isoform for their gene of interest. ! !

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Section: Introductionmentioning

confidence: 99%

Quantitative RNA-seq meta analysis of alternative exon usage in C. elegans

Tourasse

Millet

Dupuy

2017

Preprint

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“…2D, E). 8 As a second example, the Y44A mutation has further reduced RNA binding, but has comparable affinity without or with the adjacent ASD-1 (Figs. 2F,G).…”

Section: Atomic Details Of Rna Binding: Divide and Conquermentioning

confidence: 99%

“…2C) and NMR experiments demonstrate a significant mobility between the RRM domains on this ligand. 8 Another consequence of binding independence is the prediction that SUP-12 constructs with mutations in critical RNA-binding residues will display a consistent affinity in the absence or presence of ASD-1. A previous genetic screen identified a SUP-12 mutant, yb1010, where Gly113 is replaced with glutamate.…”

Section: Atomic Details Of Rna Binding: Divide and Conquermentioning

confidence: 99%

“…16 In the egl-15 model system, cooperativity would manifest itself as the increased affinity of SUP-12 for RNA when ASD-1 is also bound to the same pre-RNA molecule. Based on the series of affinity studies reported by Amrane et al, 8 the isolated RRM domain from SUP-12 binds to its minimal RNA ligand with a dissociation constant (K D ) of 69 nM ( Fig. 2A).…”

Section: Atomic Details Of Rna Binding: Divide and Conquermentioning

confidence: 99%

“…8,9 These studies highlight the steps required to determine the atomic basis of the interaction between adjacent bound splicing factors, and in this case reveals a complex balance between both favorable and unfavourable contacts between SUP-12 and ASD-1.…”

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Splicing factor SUP-12 and the molecular complexity of apparent cooperativity

Mackereth

2014

Worm

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T he splicing factor SUP-12 from C. elegans, in combination with either ASD-1 or FOX-1 from the Fox-1 (RBFOX) family, is required for generating a muscle-specific isoform of the fibroblast growth factor receptor EGL-15. Biophysical techniques have revealed the sequence preference for the RNA Recognition Motif (RRM) domain from SUP-12 as well as the structural details of the RNA-bound complex. Detailed genetics have identified a requisite need for the presence of both SUP-12 and ASD-1/FOX-1 to regulate the alternative splicing event, prompting speculation of a cooperative mechanism between these proteins on binding RNA. In contrast, the interplay between SUP-12 and ASD-1 suggests that although the RRM domains from each protein are in direct contact on the egl-15 pre-mRNA, there is no simple contribution of binding cooperativity. Evidence for an independent binding mechanism by SUP-12 and ASD-1 will be discussed, including a model in which both positive and negative contributions are balanced during complex assembly. The ability to monitor tissue-specific alternative splicing in live nematodes will continue to provide a powerful method to test in vivo mechanistic models derived from atomic-level investigation.Interactions between cellular biomolecules such as proteins and nucleic acids are critical for life, and the precise regulation of these interactions in a time-and location-dependent manner allows for normal organism development.Alternative splicing can contribute to this process through the production of development-and tissuespecific protein isoforms. The spliceosome generally interacts with target premRNA with the help of constitutive protein trans factors that recognize RNA cis elements in order to define the intron boundaries and dictate the series of exons in the mature mRNA. To regulate this process, additional splicing factors also bind to the pre-mRNA in order to enhance or inhibit specific 5 0 and 3 0 intron splice sites. The mechanisms by which the final splice patterns are established are in general poorly understood, but predominantly involve cooperative or competitive interactions with the constitutive splicing machinery to alter spliceosome recruitment, premRNA motif availability or the catalytic efficiency.Several splicing factors have already been identified in C. elegans that display limited expression patterns and lead to cell type-specific changes in splicing regulation.1 In many cases, multiple factors are linked to a specific splicing event, including both positive and negative regulators that together dictate the final pattern of exon retention in the mature mRNA. 2Structures of RNA-binding domains from splicing proteins shed light on the basis for RNA motif preference and this information can explain the phenotypic effects of known genetic variations in the protein factors or RNA elements. The molecular details also help in the strategic design of mutants to selectively perturb the contribution of a splicing factor in a given splicing event. Less known are the atomic details that under...

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An emerging model organism Caenorhabditis elegans for alternative pre‐mRNA processing in vivo

Wani

Kuroyanagi

2017

WIREs RNA

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A nematode Caenorhabditis elegans is an intron-rich organism and up to 25% of its pre-mRNAs are estimated to be alternatively processed. Its compact genomic organization enables construction of fluorescence splicing reporters with intact genomic sequences and visualization of alternative processing patterns of interest in the transparent living animals with single-cell resolution. Genetic analysis with the reporter worms facilitated identification of trans-acting factors and cis-acting elements, which are highly conserved in mammals. Analysis of unspliced and partially spliced pre-mRNAs in vivo raised models for alternative splicing regulation relying on specific order of intron excision. RNA-seq analysis of splicing factor mutants and CLIP-seq analysis of the factors allow global search for target genes in the whole animal. An mRNA surveillance system is not essential for its viability or fertility, allowing analysis of unproductively spliced noncoding mRNAs. These features offer C. elegans as an ideal model organism for elucidating alternative pre-mRNA processing mechanisms in vivo. Examples of isoform-specific functions of alternatively processed genes are summarized. WIREs RNA 2017, 8:e1428. doi: 10.1002/wrna.1428 For further resources related to this article, please visit the WIREs website.

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Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition

Cited by 21 publications

References 52 publications

Quantitative RNA-seq meta analysis of alternative exon usage in C. elegans

Quantitative RNA-seq meta analysis of alternative exon usage in C. elegans

Splicing factor SUP-12 and the molecular complexity of apparent cooperativity

An emerging model organism Caenorhabditis elegans for alternative pre‐mRNA processing in vivo

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