A novel protein domain in an ancestral splicing factor drove the evolution of neural microexons

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The mechanisms by which entire programmes of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates, and are critical for proper brain development and function. Here, we discover neural microexon programmes in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized ‘enhancer of microexons’ (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralogue Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programmes that qualitatively expanded the neuronal molecular complexity in bilaterians.

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Fig. 1: Neural microexon programmes in bilaterian animals.
Fig. 2: Evolution of the Srrm2/3/4 locus in metazoans.
Fig. 3: The eMIC domain is key for neural microexon splicing.
Fig. 4: The eMIC domain interacts with early spliceosomal factors to promote microexon inclusion.

Code availability

All of the software used to analyse the data is publicly available and listed in the Reporting Summary. VastDB files to run vast-tools are available to download for each species (https://github.com/vastgroup/vast-tools), as indicated in the Methods. Custom codes to generate orthologous gene clusters and figure plots are available upon request.

Data availability

Raw RNA-Seq data were submitted to the Sequence Read Archive (SRP149913). Mass spectrometry data were submitted to the ProteomeXchange Consortium: AP-MS through MassIVE (https://massive.ucsd.edu; accession codes: MSV000082361 and PXD009779) and RNP mass spectrometry via PRIDE59 (PXD010034). All other RNA-Seq datasets used in the study are listed in Supplementary Dataset 3.


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The authors thank B. Lehner and S. W. Roy for critical reading of the manuscript, M. Akam and K. Siggens for providing access to Strigamia specimens, M. Sattler and P. Zou for pETM11 clones, S. Taylor for providing the HeLa Flp-In cell line, B. Bergum (Flow Cytometry Core Facility at the University of Bergen) for assistance with Nematostella fluorescence-activated cell sorting, and the CRG Genomics Unit. Animal silhouettes were obtained from PhyloPic. This work has been funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-StG-LS2-637591 to M.Ir. and ERC-AdvG-670146 to J.V.), the Spanish Ministry of Economy and Competitiveness (BFU2014-55076-P and BFU2017-89201-P to M.Ir., BFU2014-005153 to J.V., and the ‘Centro de Excelencia Severo Ochoa 2013–2017’ (SEV-2012-0208)), AGAUR, Fundación Botín (to J.V.) and the Canadian Institutes of Health Research (to B.J.B. and A.-C.G.). RNP mass spectrometric analyses were performed at the CRG/UPF Proteomics Unit (part of ProteoRed-PRB3, supported by PE I+D+i 2013–2016 (PT17/0019) of the ISCIII and ERDF) by ‘Programa CERCA Generalitat de Catalunya’ and ‘Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement’ (2017SGR595). A.T.-M. held an FPI-SO fellowship, and Y.M. a Marie Skłodowska-Curie individual fellowship.

Author information

A.T-M. performed the bioinformatic analyses and molecular biology and cell culture experiments. S.B. performed the cell culture, spliceosome assembly and RNP mass spectrometry experiments under the supervision of J.V. Y.M. performed the bioinformatics analyses. J.R. performed and analysed the AP-MS experiments under the supervision of A-C.G. and B.J.B. M.Ig. performed the Nematostella experiments under the supervision of F.R. J.P. performed the Strigamia dissections and molecular biology work. D.O. performed the co-immunoprecipitation experiments under the supervision of B.J.B. T.G., I.A., F.G. and M.S. performed the Drosophila and molecular biology experiments. M.Ir. conceived and supervised the study, provided resources and performed the bioinformatic analyses. A.T-M., S.B., M.Ir. and B.J.B. wrote the manuscript with input from all authors.

Correspondence to Manuel Irimia.

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Supplementary information

Supplementary Information

Supplementary Figures 1–7

Reporting Summary

Supplementary Data 1

Conserved microexons across phyla

Supplementary Data 2

Mass spectrometry results

Supplementary Data 3

RNA-seq data used in this study

Supplementary Data 4

Orthologous genes in the six bilaterian species studied

Supplementary Data 5

Srrm protein sequences used for comparative analyses

Supplementary Data 6

List of primer sequences used in this study

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