Role of the SAF-A/HNRNPU SAP domain in X chromosome inactivation, nuclear dynamics, transcription, splicing, and cell proliferation
Judith A. Sharp, Emily Sparago, Rachael Thomas, Kaitlyn Alimenti, Wei Wang, Michael D. Blower
Abstract
SAF-A/HNRNPU is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. Here we show the SAF-A protein is highly dynamic and interacts with nascent transcripts as part of this dynamic movement. This finding revises current models of SAF-A: rather than being part of a static nuclear scaffold/matrix structure that acts as a stable tether between RNA and chromatin, SAF-A executes nuclear functions as a dynamic protein, suggesting contacts between SAF-A, RNA, and chromatin are more high turnover interactions than previously appreciated.
Introduction
Recent work has shown that RNA plays an active role in forming compartments in the nucleus to regulate various essential functions, such as RNA processing and gene expression [1–5]. RNA and RNA-binding proteins are concentrated near active chromatin and act to organize a network of transcription factors and other chromatin regulators. The proper assembly of these structures is therefore important for essential nuclear functions and to prevent neurological disease [6,7].
Materials and method
Cell culture and drug treatment
hTERT-immortalized RPE-1 cells were a gift from Brian Chadwick (Florida State University, Tallahassee, FL; ATCC CRL-4000) and were cultured as described [27]. The SAF-A degron cell line in RPE-1 cells has been described [27]. Lentiviruses with SAF-Awt-GFP, SAF-AAA-GFP, SAF-ADD-GFP, and SAF-AΔSAPGFP were prepared and used to transduce the SAF-A-degron cell line. Individual clones were expanded and validated by immunofluorescent detection of GFP and western blot analysis.
Results
The SAF-A SAP domain is required for cell proliferation
To investigate SAF-A nuclear functions in human cells, we first designed a cell line to degrade endogenous SAF-A in diploid, karyotypically stable human RPE-1 cells immortalized with hTERT ( [27]; Fig 1B). Both SAF-A genes were modified at the 3’ end to include the minimal auxin-inducible degron sequence and a mCherry fluorescent tag (SAF-A-AID-mCherry). A doxycycline-inducible TIR1 gene encoding the E3 ligase required for auxin-mediated protein degradation was integrated by lentivirus.
Discussion
In this study, we describe how a degron system in diploid, karyotypically stable RPE-1 cells can be used to explore SAF-A function in several genetic assays. Given the high abundance of SAF-A in the nucleus [59], one advantage of this system is that it allows for the rapid depletion of SAF-A, allowing for observation within 24 hours, as well as longer time periods. This allowed us to obtain time resolved datasets for the role of SAF-A in cell proliferation, gene expression, and splicing.
Acknowledgments
The authors thank Reito Watanabe for helpful suggestions. We thank Chi Jing and Nelson Lau for preparation of the RPE-1 genome sequencing library. We thank Daniel Cifuentes for use of the LiCor fluorescent imager. We acknowledge that much of the computational work reported on in this paper was performed on the Shared Computing Cluster which is administered by Boston University’s Research Computing Services.
Citation: Sharp JA, Sparago E, Thomas R, Alimenti K, Wang W, Blower MD (2025) Role of the SAF-A/HNRNPU SAP domain in X chromosome inactivation, nuclear dynamics, transcription, splicing, and cell proliferation. PLoS Genet 21(6): e1011719. https://doi.org/10.1371/journal.pgen.1011719
Editor: Nick Gilbert, The University of Edinburgh, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: October 30, 2024; Accepted: May 8, 2025; Published: June 10, 2025
Copyright: © 2025 Sharp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All raw reads and summary files are available in GEO under the accession numbers: GSE277216, GSE277217, GSE277219, GSE277221.
Funding: This work was supported by the National Institutes of General Medical Sciences GM144352 to MB and National Institutes of General Medical Sciences GM122893 to MB. https://www.nigms.nih.gov/he funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
