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
- Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, INIBIOMA (CONICET-UNComa), Argentina
- University of Bonn, Institute of Innate Immunity, Medical Faculty, Germany
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, United Kingdom
- Edinburgh Cancer Research UK Centre, Institute of Genetics and Cancer, University of Edinburgh, United Kingdom
- Edinburgh University, 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.
Metrics
-
- 2,862
- views
-
- 356
- downloads
-
- 28
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function
eLife 10:e65104.
https://doi.org/10.7554/eLife.65104
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.
-
- Biochemistry and Chemical Biology
Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
