Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, United Kingdom
- Institute of Nuclear Technologies for Health (Intecnus), Argentina
- University of Bonn, Institute of Innate Immunity, Medical Faculty, Germany
- Edinburgh Cancer Research UK Centre, Institute of Genetics and Cancer, University of Edinburgh, United Kingdom
Abstract
Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.
Data availability
Total RNA-seq data related to splicing analysis have been deposited in GEO under accession code GSE157269G. Polysomal, monosomal and cytoplasmic RNA-sequencing data have been deposited in GEO under accession code GSE161828. The mass spectrometry proteomics data is presented as LFQ values in the form of Excel tables in Figure 4-source data 1 and in Figure 4-figure supplement 1-source data 1. The complete raw datasests can be downloaded from ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD019859. Cilia motility data analyzed in Fiji using the custom-written ImageJ plugin "Cilility_JNH" was deposited in zenodo (https://doi.org/10.5281/zenodo.5138072).
Article and author information
Author details
Magdalena M Maslon
Funding
Medical Research Council (MRC Core funding)
- Javier F Caceres
H2020 European Research Council (Consolidator grant 866355)
- Pleasantine Mill
Deutsche Forschungsgemeinschaft (Germany's Excellence Strategy)
- Dagmar Wachten
Wellcome Trust (Multiuser Equipment 208402/Z/17)
- Alex von Kriegsheim
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Douglas L Black, University of California, Los Angeles, United States
Ethics
Animal experimentation: This is stated in the Materials and Methods Section, 'Animal experiments'We followed international, national and institutional guidelines for the care and use of animals. Animal experiments were carried out under UK Home Office Project Licenses PPL 60/4424, PB0DC8431 and P18921CDE in facilities at the University of Edinburgh (PEL 60/2605) and were approved by the University of Edinburgh animal welfare and ethical review body.
Version history
- Preprint posted: September 4, 2020 (view preprint)
- Received: November 23, 2020
- Accepted: July 30, 2021
- Accepted Manuscript published: August 2, 2021 (version 1)
- Accepted Manuscript updated: August 4, 2021 (version 2)
- Accepted Manuscript updated: August 5, 2021 (version 3)
- Version of Record published: August 9, 2021 (version 4)
Copyright
© 2021, Haward et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function
eLife 10:e65104.
https://doi.org/10.7554/eLife.65104
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Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.
