Role of the SAF-A/HNRNPU ATPase and RGG domains in X chromosome inactivation, nuclear dynamics, transcription, splicing, and cell proliferation

Abstract

The SAF-A/HNRNPU gene encodes an abundant nuclear protein conserved throughout vertebrates, and is mutated in individuals with HNRNPU syndrome, a neurological human disease. SAF-A is important for maintaining lncRNA localization, splicing, and gene expression state. The mechanistic role of SAF-A in each of these processes is coordinated by one or more of its functional domains, which include an N-terminal SAP domain, a central ATPase domain, and a series of C-terminal RGG repeats embedded in a low-complexity region. The SAP domain and RGG repeats define two nucleic acid interaction domains, with both capable of binding DNA or RNA. Here we use an allelic reconstitution strategy to investigate the role of the SAF-A ATPase domain and RGG repeats. We show that both the ATPase and RGG repeats control SAF-A nuclear dynamics, and present genetic evidence that SAF-A interacts with nascent transcripts through the RGG repeats. The SAF-A ATPase domain and RGG repeats were also required for maintaining XIST RNA and facultative heterochromatin marks on the inactive X chromosome, with distinct effects of mutations that block ATP binding and ATP hydrolysis. Analysis of transcriptome datasets revealed that the SAF-A ATPase domain and RGG repeats are both required for proper mRNA splicing, but not for gene expression. Importantly, we found that like the SAP domain, the SAF-A ATPase domain and RGG repeats are required for cell proliferation. Collectively, our findings highlight the importance of the SAF-A SAP, ATPase and RGG domains in essential functions of nuclear biology. These analyses will therefore inform our understanding of the disease state in HNRNPU syndrome.

Figure 1.. The ATPase and RGG domains are required for cell proliferation.

A. Schematic depicting full-length SAF-A (isoform a, 825 amino acids) with domains drawn to scale. The position of the seven RGG repeats is marked between residues 702–775. B. Design of the SAF-A-AID-mCherry degron to replace the endogenous SAF-A genes in RPE-1 cells. C. SAF-A-GFP tagged transgenes used to reconstitute either wild-type SAF-A, or versions of SAF-A bearing point mutations in the Walker A or Walker B elements in the ATPase domain, or a deletion of residues 702–775 containing RGG repeats 1–7. D. Western blot analysis of SAF-A cell lines to monitor expression of SAF-A-AID-mCherry (α-RFP panel) and SAF-A-GFP (α-GFP panel) in cell lines. Tubulin was used as a loading control. Lane 1: untagged RPE-1 parental cell line. Lane 2: SAF-A degron cell line, either without (lane 2), or with (lane 3) doxycycline and IAA. Lanes 4–7: cells expressing SAF-A-GFP transgenes, treated with doxycycline and IAA. Molecular weight markers are indicated to the right of the panel. E. Immunofluorescence of cells expressing tagged SAF-A, as indicated by the panel insets. Treatment with doxycycline and IAA as indicated led to complete depletion of SAF-A-AID-mCherry in the vast majority of cells. Integrated SAF-A-GFP transgenes showed uniform expression in the cell population after induction. Bar, 40 μm. F-G. PCNA and Ki-67 immunofluorescence to monitor cell proliferation. Cells were treated with doxycycline and IAA to induce endogenous SAF-A depletion and SAF-A-GFP transgene expression, and were compared to control cell populations. Cells were treated for 1, 3, 6, or 10 days as indicated. SAF-A expression is the same as the panel insets shown in E. All images in panels E-G are rendered as a maximum projection of a 3D stack. H. Quantitation of cell populations with PCNA- and Ki-67-positive cells expressed as percent of the total population. 300 cells were scored for PCNA and Ki-67 immunofluorescence in two biological replicates; the average and standard deviation is shown.

Figure 2.. The SAF-A RGG and ATPase domains are important for nuclear dynamics.

A. Images from a typical FRAP experiment using SAF-A^wt-GFP. B. Table of the t^1/2 recovery time and the immobile fraction for all SAF-A-GFP proteins, with and without transcriptional inhibition. C-F. FRAP recovery curves for SAF-A^wt-GFP, SAF-A^ΔWalker-A-GFP, SAF-A^ΔWalkerB-GFP, and SAF-A^ΔRGG-GFP, with and without transcriptional inhibition (LDC). The standard deviation of recovery time is indicated in light colored error bars. G. SAF-A^wt-GFP, SAF-A^ΔWalker-A-GFP, SAF-A^ΔWalkerBGFP, and SAF-A^ΔRGG-GFP were immunoprecipitated and co-precipitated RNAs were sequenced and normalized to Drosophila spike-in control RNA. Enrichment of RNA in all mutants was compared to WT and is plotted as a Cumulative Distribution Function.

Figure 3.. Recruitment of SAF-A to the Xi is independent of the ATPase and RGG domains.

A-F. Live cell analysis of SAF-A^wt-GFP and SAF-A domain mutations as indicated. Two examples of SAF-A^{ΔRGG 1–7}-GFP are shown. Cells were analyzed 24 hours after doxycycline and IAA treatment. Each image represents a single 0.2 mm slice. Bar, 10 μm. The proportion of cells showing the depicted localization patterns for each allele are as follows: SAF-A^wt-GFP, 97% of cells; SAF-A^ΔSAP -GFP, 100% of cells; SAF-A^{ΔWalker A}-GFP 100% of cells; SAF-A^{ΔWalker B}-GFP 99% of cells; SAF-A^{ΔRGG 1–7}-GFP 100% of cells (n ≥ 50). At least two independent live imaging experiments for each genotype were performed.

Figure 4.. The ATPase domain and RGG 1–7 repeats impact XIST RNA localization and PRC1/PRC2-dependent histone modifications on the Xi.

A. XIST RNA FISH and DAPI staining in RPE-1 cells, and cells expressing SAF-A-GFP transgenes, 24 hours after treatment with doxycycline and IAA. Images are rendered as a maximum projection of a 3D stack. Bar, 10 μm. B. Quantitative measurement of XIST RNA foci per cell. The term foci refers to the number of resolvable objects rather than individual molecules. Image stacks were acquired for at least 100 nuclei per genotype for two biological replicates and analyzed in Fiji software to count XIST RNA foci. Measurements are depicted as violin superplots. The average of each replicate is depicted as an open circle, whereas the average of both replicates is depicted as a horizontal line. The standard deviation of the two averages is shown. Cell n for quantitation of XIST RNA particles is: RPE-1 n = 193 and n = 211, SAF-A^wt-GFP n = 137 and n = 236, SAF-A depleted 1 n = 122 and n = 220, SAF-A^ΔWalker-A-GFP n = 135 and n = 193, SAF-A^{ΔWalker B}-GFP n = 177 and n = 183, SAF-A^{ΔRGG 1–7}-GFP n = 159 and n = 180. Statistical comparison of number of XIST RNA particles was performed using a one-way ANOVA followed by Tukey’s tests with a Bonferroni correction. The p-value comparisons between SAF-A^wt and all other genotypes are as follows: SAF-A^wt versus SAF-A^wt-GFP, ns. SAF-A^wt versus SAF-A depleted, p = 0.001. SAF-A^wt versus SAF-A^ΔWalker-A-GFP, 0.088. SAF-A^wt versus SAF-A^{ΔWalker B}-GFP, p = 0.003. SAF-A^wt versus SAF-A^{ΔRGG 1–7}-GFP, p = 0.001. C. Immunofluorescence of histone modifications H2AK119ub and H3K27me3 in RPE-1 cells and SAF-A depleted cells, 24 hours after treatment with doxycycline and IAA. Nuclei were stained with DAPI. Merged images are rendered as a maximum projection of a 3D stack. Bar, 10 μm. D. Quantitation of cell populations with H2AK119ub and H3K27me3 enrichment on the Xi. 100 cells were scored in two biological replicates; the average and standard deviation is shown. Statistical comparison was performed using a t-test to determine p-values for H2AK119ub enrichment. SAF-A^wt versus SAF-A^wt-GFP, p = 0.860, ns. SAF-A^wt versus SAF-A^ΔWalker-A-GFP, p = 0.380, ns. All other comparisons were statistically significant: SAF-A^wt versus SAF-A depleted, p = 0.007. SAF-A^wt versus SAF-A^{ΔWalker B}-GFP, p = 0.028. SAF-A^wt versus SAF-A ^{ΔRGG 1–7}-GFP, p = 0.003. The same analysis was performed to determine p-values for H3K27me3 enrichment. SAF-A^wt versus SAF-A^wt-GFP, p = 0.870, ns. All other comparisons were statistically significant: SAF-A^wt versus SAF-A depleted, p = 0.002. SAF-A^wt versus SAF-A^ΔWalker-A-GFP, p = 0.004. SAF-A^wt versus SAF-A^{ΔWalker B}-GFP, p = 0.001. SAF-A^wt versus SAF-A^{ΔRGG 1–7}-GFP, p = 0.001. All images shown are projections of an optical stack of 0.2 μm slices.

Figure 5.. SAF-A depletion leads to widespread changes in mRNA splicing.

A. mRNA splicing was evaluated in SAF-A depleted cells (24 hours) using rMATS. Upset plot compares the intersection of all significantly misspliced exons between each condition. B. Histogram plot showing changes in exon inclusion in each SAF-A mutant and the SAF-A depleted cells. C. PCA plot comparing each SAF-A mutant for all common exons assayed. D. CDF plot showing the magnitude of splicing changes for exons that are significantly altered in SAF-A depleted cells for each of the mutants plotted. E. Sashimi plot showing an example altered exon in each mutant.