FUS regulates a subset of snoRNA expression and modulates the level of rRNA modifications

Abstract

FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2'-O methylation (2'-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2'-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2'-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.

Figure 1

Volcano plots for proliferating (A) and differentiated (B) SH-SY5Y cells. Enhanced Volcano plots were generated using Enhanced Volcano R package. Raw count matrix with only snoRNAs was used in DESeq2 to get differentially expressed snoRNAs (padj ≤ 0.05), the tabular file generated was then used in Enhanced Volcano library with x-axis = 'log2FoldChange' and y-axis = 'padj'. Representative differentially expressed snoRNAs are annotated. (NS: non-significant).

Figure 2

Differential heatmap for variations in 2’-O-Me levels in all cells analyzed, representing the mean of three biological replicates from each cell line (A). Heatmap was generated using Graphpad Prism 8.4.2 (https://www.graphpad.com/scientific-software/prism/). Heatmap represents sites with at least a 10% (0.1) difference in 2’-O-Me between cell types. RT-qPCR analysis of relative expression levels of selected C/D box snoRNAs that guide methylation of residues in 18S rRNA and 28S rRNA (B). The values represent the mean and error bars indicate the SD of three biological replicates from each cell line. HEK293T FUS KO cells, proliferating (Proli) SH-SY5Y FUS KO, differentiated (Diff) SH-SY5Y FUS KO cells, and SH-SY5Y FUS R495X cells were compared to WT cells, respectively. P values were calculated using Student’s t-test, and the statistical significance is defined as follows: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Small RNA-seq results for some of the differentially expressed C/D box snoRNAs identified by DEseq2 analysis in Proli and Diff SH-SY5Y FUS KO cells and presented as relative fold change as compared to WT cells (C). *Padj ≤ 0.05; **Padj ≤ 0.01; ***Padj ≤ 0.001.

Figure 3

Scheme representing positions in 18S and 28S rRNAs with sites of methylation and pseudouridylation differentially modified in FUS KO and FUS R495X cells in comparison to wild-type cells. Red, blue and white bars indicate upregulation, downregulation or no changes, respectively. snoRNAs responsible for modifications are located above corresponding sites, red and blue arrows indicate snoRNA upregulation or downregulation, respectively, confirmed by small RNA-seq or RT-qPCR.

Figure 4

Differential heatmap for variations in pseudouridylation levels in all cells analyzed, representing the mean of three biological replicates from each cell line (A). Heatmap was generated using Graphpad Prism 8.4.2 (https://www.graphpad.com/scientific-software/prism/). Heatmap represents sites with at least a 10% (0.1) difference in pseudouridylation between cell types. RT-qPCR analysis of relative expression levels of selected box H/ACA snoRNAs that guide pseudouridylation of residues in 18S rRNA and 28S rRNA (B). The values represent the mean and error bars indicate the SD of three biological replicates from each cell line. HEK293T FUS KO cells, proliferating (Proli) SH-SY5Y FUS KO cells, differentiated (Diff) SH-SY5Y FUS KO cells, and SH-SY5Y FUS R495X cells were compared to WT cells, respectively. P values were calculated using Student’s t-test, and the statistical significance is defined as follows: *P ≤ 0.05. Small RNA-seq results for some of the differentially expressed H/ACA box snoRNAs identified by DEseq2 analysis in Diff SH-SY5Y FUS KO cells and presented as relative fold change as compared to WT cells (C). *Padj ≤ 0.05; ***Padj ≤ 0.001.

Figure 5

RNA immunoprecipitation (RIP) experiment to test whether FUS can bind C/D box snoRNAs (A), H/ACA box snoRNAs (B), and snoRNA-host gene transcripts (C) in HEK293T cells. 7SL RNA was used as a negative control, U1 snRNA and U7 snRNAs were used as a positive controls (D). Arrows next to snoRNA names indicate the direction of changes in the expression of hosted snoRNAs in FUS KO cells relative to WT cells. Bars represent the mean and error bars indicate the SD of three biological replicates. P values were calculated using Student’s t-test, and the statistical significance is defined as follows: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 6

Visualization of the 2’-O-Me and pseudouridylation sites with the significant differences in FUS KO and FUS R495X cells on the LSU (28S, 5.8S, and 5S rRNA) and SSU (18S rRNA). Ribbon representation of PDB:4UG0 file. All the rRNAs are denoted by a unique colored ribbon and labelled in the same color (A, C and E). Representation of only modified residues, green for 2’-O-Me and magenta for pseudouridylation with only 5.8S rRNA in a red ribbon form to show structure orientation. Modified sites in SSU (A and B), modified sites in LSU (C and D), modified sites on SSU and LSU combined (E and F). uS19 is represented as a green color cartoon, with ARG10 denoted as brown-colored spheres. *B1a is an inter-subunit bridge formed by the interaction between ARG10 of uS19 and Psi-1779. DCS, decoding center (SSU), PTC, peptidyl transferase center (LSU). This figure was generated using published human 80S ribosome structure, 4UGO. The illustration was done using PyMol2 (https://pymol.org/2/).