SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells
- Rachel E. Cherney1,2,3,4,
- Quinn E. Eberhard1,2,3,5,9,
- Gilbert Giri1,2,3,5,9,
- Christine A. Mills1,6,9,
- Alessandro Porrello2,3,9,
- Zhiyue Zhang7,8,9,
- David White7,8,
- Jackson B. Trotman1,2,3,
- Laura E. Herring1,6,
- Daniel Dominguez1,2,3 and
- J. Mauro Calabrese1,2,3
- 1Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 2RNA Discovery Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 4Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 5Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 6Proteomics Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 7Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- 8Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- Corresponding author: jmcalabr{at}med.unc.edu
-
↵9 These authors contributed equally to this work.
Abstract
Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1, which also contains a purine-rich region in its 5′ end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA.
Keywords
- Received December 21, 2022.
- Accepted June 23, 2023.
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