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. 2024 Dec 31;147(2):1458–1462. doi: 10.1021/jacs.4c17659

Quantitative Proteomics Identifies Profilin-1 as a Pseudouridine-Binding Protein

Songbo Wei †, Xiaoxia Dai †, Jun Yuan †, Shiyang He ‡, Kriti Shah ‡, Shiyuan Guo §, Zheng Duan †, Jernej Murn ‡, Yinsheng Wang †,§,*
PMCID: PMC11744752  PMID: 39812085

Abstract

graphic file with name ja4c17659_0003.jpg

Pseudouridine (Ψ) is the most abundant RNA modification in nature; however, not much is known about the biological functions of this modified nucleoside. Employing an unbiased quantitative proteomics method, we identified multiple candidate reader proteins of Ψ in RNA, including a cytoskeletal protein profilin-1 (PFN1). We demonstrated that PFN1 binds directly and selectively to Ψ-containing RNA. Additionally, we discovered approximately 4000 binding sites of PFN1 in human cells, including a known dyskerin (DKC1)-installed Ψ site in TPI1 mRNA, which encodes triosephosphate isomerase. Moreover, we showed that PFN1 and DKC1 are crucial for regulating the stability and translation efficiency of TPI1 mRNA through modulating PFN1-Ψ interaction. Together, we identified PFN1 as a reader protein of Ψ in RNA and illustrated a potential role of PFN1-Ψ interaction in post-transcriptional regulation. These findings provide new insights into the functions of Ψ in RNA biology and in modulating the expression of an important metabolic enzyme.

RNA harbors over 170 chemically distinct modified residues,1 which play vital roles in various aspects of RNA biology.2 Pseudouridine (Ψ) is the most abundant modified nucleoside in RNA, and is present in all major types of RNA,3−7 impacting various biological processes and disease pathogenesis.2,8 The importance of Ψ is also manifested by the success of the COVID-19 mRNA vaccine, which is modified with N1-methyl-pseudouridine.9 Ψ can be installed by 13 well-annotated Ψ synthases (“Ψ writers”) in human cells;6,10,11 relatively little, however, is known about the reader proteins or the biological functions of Ψ.2,5,6,10,12

Profilin-1 (PFN1) is a cytoskeletal protein interacting with G-actin,13 poly-l-proline-containing ligands, phosphoinositide lipids, and microtubules14 to regulate various important cellular processes.15 Mutations in this protein have been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS),15 where the ALS-linked PFN1 variants can negatively impact RNA processing.16 Previous studies identified PFN1 as a candidate RNA-binding protein; however, it remains unknown how PFN1 recognizes RNA.17,18

Here, we employed a quantitative mass spectrometry-based method to identify proteins that can bind to Ψ in RNA. Our results revealed multiple candidate Ψ-binding proteins, including PFN1, which we demonstrated to be capable of binding directly to Ψ-containing RNA. We also showed that PFN1 and DKC1 are crucial for regulating the stability and translation efficiency of TPI1 mRNA through modulating PFN1-Ψ interaction.

To better understand the biological functions of Ψ in RNA, we employed stable isotope labeling by amino acid in cell culture (SILAC)-based quantitative proteomics to systematically screen cellular proteins that can bind to Ψ-containing RNA. We used a 5′-biotin-labeled Ψ-containing RNA derived from human TERC mRNA, which was shown to harbor two Ψ residues in HEK293T cells,4,19 as the probe bait and the corresponding uridine-containing RNA as the control. To eliminate potential experimental bias, we carried out both forward and reverse SILAC experiments (Figure 1a). Our results revealed multiple proteins displaying preferential binding to Ψ-containing RNA over the corresponding U-containing RNA (Figure 1b). Many of these proteins were shown to interact with RNA, including YTHDF120 and several metabolic enzymes (Figure 1b, Table S2). Moreover, cytoskeletal protein PFN1 was enriched only by the Ψ-containing RNA probe, indicating its preferential binding for Ψ over U in RNA (Figures 1b-d, S1).

Figure 1.

Figure 1

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PFN1 is a Ψ-binding protein. (a) A schematic diagram illustrating the forward SILAC workflow. (b) Quantitative proteomics identified multiple candidate Ψ-binding proteins. (c, d) Representative ESI-MS for the [M + 2H]2+ ions of a tryptic peptide of PFN1, STGGAPTFNVTVTK, acquired from forward and reverse SILAC experiments. (e) Normalized anisotropy of PFN1 in binding with Ψ- and U-containing RNA. Error bars represent SD (n = 3). (f) The Kd values for the binding of PFN1 with Ψ- and U-containing RNA. (g) LC-MS/MS data showing the enrichment of Ψ-containing RNA by PFN1 (comparing PFN1-bound fractions, i.e., RIP and CLIP, with input and flow-through samples, i.e., RIP-FT and CLIP-FT). **, 0.001 ≤ p < 0.01; ***, p < 0.001 (unpaired, two-tailed Student’s t-test).

We next examined whether PFN1 can bind directly Ψ-containing RNA. We purified recombinant human PFN1 and used fluorescence anisotropy to measure its binding affinities for the Ψ- and the corresponding U-containing RNA (Figure 1e-f). We found that PFN1 binds more strongly to Ψ- than U-containing RNA (Kd = 96 ± 13 μM versus 298 ± 57 μM; Figure 1e-f). It is worth noting that our SILAC data revealed a more pronounced selectivity of PFN1 in binding toward the Ψ- over the U-bearing RNA (Figures 1b-d, S1), indicating that formation of protein complex(es) may enhance PFN1’s binding to Ψ-containing RNA. The physiological concentration of PFN1 protein is approximately 121 μM in N2a cells,21 suggesting that the interaction of PFN1 with Ψ-containing RNA is physiologically relevant. In addition, PFN1 is known to form cellular condensate,22 where multivalent interactions may further augment the interaction of PFN1 with Ψ-containing RNA. We also incubated recombinant PFN1 protein with total RNA isolated from cells and extracted PFN1-bound RNA from the mixture, digested it to mononucleosides, and subjected the digestion mixture to LC-MS/MS analysis. Our results showed that Ψ/U ratio is significantly higher in the pulldown samples than the input and flow-through samples (Figures 1g, S2 & S3). Notably, cross-linking with 254 nm light prior to the PFN1 isolation further increased the Ψ/U ratio in the PFN1-bound RNA (Figure 1g). Together, these results showed that PFN1 can bind directly and preferentially to Ψ-containing RNA.

Next, we employed iCLIP23 for transcriptome-wide mapping of PFN1-RNA interactions in human cells (Figure S4a-c). Our results revealed approximately 4000 PFN1 binding sites, which spread across various types of RNA, including mRNAs and noncoding RNA (ncRNA, Figure S4d), and are consistent with the widespread presence of Ψ in these RNA subtypes.3−7,24 For example, our data showed strong PFN1 iCLIP signals in RNU6-9 and SOX2-OT, where multiple Ψ sites have been detected10 (Figure S5a,b). Consistent with the observations that mRNA also harbors a considerable proportion of Ψ modifications,6,10,25 we found that hundreds of PFN1 binding sites are located in mRNA (Figures S4d and S5c,d). Additionally, PFN1 binding sites overlap with known Ψ sites at multiple positions across different mRNAs (e.g., hnRNPC and PARP6)25 (Figure S5c,d, Table S3). Notably, we identified a PFN1 binding site that overlaps with a DKC1-installed Ψ site in the mRNA of TPI1 gene,10,25 which encodes triosephosphate isomerase and is implicated in neurodegenerative diseases26,27 (Figure 2a, Table S3).

Figure 2.

Figure 2

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PFN1 binds to a DKC1-installed Ψ in TPI1 mRNA, whose stability and translation efficiency are regulated by PFN1 and DKC1. (a) A PFN1 binding site overlaps with a known DKC1-installed Ψ site in TPI1 mRNA. A red solid arrow denotes the Ψ site. *a: ELAP-Seq,25 *b: PRAISE.10 (b-c) Depletion of PFN1 and/or DKC1 reduced TPI1 mRNA half-life (b) and promoted TPI1 protein translation (c). Error bars represent SD (n = 3). **, 0.001 ≤ p < 0.01; ***, p < 0.001 (unpaired, two tailed Student’s t-test).

Interestingly, gene ontology (GO) analysis showed that PFN1 binds to transcripts coding for proteins involved in RNA binding (Figure S6). We also found multiple significantly enriched motifs (Figure S7, Table S4). Our PFN1 iCLIP data uncovered 22 binding sites overlapping with known Ψ sites (Table S3 and Figure S8).6,10,25 Using bedtools and R to shuffle PFN1 binding sites across the expressed transcripts based on a ribo-minus RNA-seq data of HEK293T cells28 for 1,000 iterations, we found that the overlaps between PFN1 binding sites and Ψ sites from ELAP-seq and PRAISE data sets are significant (Table S5). It is of note that current Ψ-mapping methods may not capture all bona fide Ψ sites. As Ψ-mapping technologies advance, more PFN1 binding sites identified here may overlap with newly uncovered Ψ sites (e.g., a 10-fold increase in overlap observed for ELAP compared to PRAISE, Figure S8).10,25

Importantly, the Ψ site in the TPI1 mRNA to which PFN1 binds is installed by DKC1,10 a major Ψ synthase.4 We performed bisulfite/sulfite-induced deletion followed by next-generation sequencing6,10 to assess the change in modification frequency at the identified Ψ site in TPI1 mRNA in HEK293T cells upon DKC1 knockdown. Our result showed a substantial decrease in deletion ratio (from 93% to 62%) in HEK293T cells upon DKC1 knockdown (Figure S9). To validate the specific binding of PFN1 at the identified Ψ site, we conducted CLIP-qPCR to assess the binding of PFN1 with TPI1 mRNA in HEK293T cells treated with control siRNA or siDKC1. We observed a significantly diminished enrichment of TPI1 mRNA in PFN1 CLIP samples prepared from DKC1-depleted cells, substantiating that the interaction depends on DKC1 (Figure S10). Furthermore, in vitro binding experiments revealed that pseudouridylation enhanced substantially the binding of both Flag-tagged and untagged PFN1 with an oligoribonucleotide derived from the PFN1-binding site of the TPI1 mRNA (Figure S11).

Pseudouridylation can increase the stability of synthetic mRNA.29 Moreover, Ψ installed by TRUB1, another major Ψ writer, stabilizes the target mRNAs.6 Hence, incorporation of Ψ into mRNAs may enhance their stabilities.6,29 To test this, we measured the half-life (t1/2) of the TPI1 mRNA in HEK293T cells incubated with control siRNAs or siRNAs targeting PFN1 and/or DKC1. We found that the t1/2 of the TPI1 mRNA was decreased upon PFN1 and/or DKC1 depletion (Figure 2b). Notably, dual depletions of PFN1 and DKC1 did not lead to significant additional decrease in t1/2 for TPI1 mRNA, suggesting that these two proteins modulate the stability of TPI1 mRNA through the same pathway.

To further examine the role of PFN1-Ψ interaction in modulating the stability of TPI1 mRNA, we engineered a pRK7-Flag-TPI1 plasmid with the Ψ site involved in PFN1 binding being mutated to a cytosine, which abolishes the conversion of U to Ψ at this site. We observed that the t1/2 for wild-type Flag-TPI1 mRNA was significantly attenuated upon siRNA knockdown of PFN1 and DKC1; the t1/2 for the mutated Flag-TPI1 mRNA was, however, not altered upon depletion of PFN1 or DKC1 (Figure S12). Thus, these results are consistent with a role of site-specific pseudouridylation and its recognition by PFN1 in influencing TPI1 mRNA stability, though we cannot formally exclude the possibility that other factors induced by the U-to-C substitution (e.g., change in RNA conformation) might also contribute, in part, to the observed effects.

The presence of Ψ in protein-coding regions was reported to reduce protein production,30 possibly through impeding translation elongation.31 In fact, mRNAs with fewer Ψ sites due to knockdown of DKC1 are more efficiently translated.32 Consistently, we observed an augmented TPI1 protein level in HEK293T cells depleted of DKC1 (Figure 2c and Figure S10a). Knockdown of PFN1, alone or together with knockdown of DKC1, led to elevated levels of TPI1 protein in HEK293T cells (Figure 2c). In this context, it is worth noting that protein expression is not always correlated with mRNA expression.33 While the exact mechanisms through which PFN1-Ψ interaction alters mRNA stability and translation efficiency await further investigation, our results support that disruptions of this interaction through genetic depletion of the reader (PFN1) and writer (DKC1) proteins of Ψ exert the same effects on the stability of TPI1 transcript. Likewise, such disruptions forge similar effects on the translation of TPI1 mRNA.

It is worth noting that our proteomic data also revealed TPI1 as a candidate Ψ-binding protein (Figure 1b). It will be important to examine, in the future, whether TPI1 is also capable of binding preferentially with Ψ-bearing RNA, and, if so, how the binding may modulate the stability and translation efficiency of TPI1 mRNA.

Ψ is the most abundant RNA modification in nature.2 While there are 13 annotated Ψ synthases2 and remarkable advances have been made to map Ψ at single-nucleobase resolution,6,10,34,35 the exact biological functions of Ψ remain elusive, mostly owing to the lack of known reader proteins.12 So far, few Ψ readers have been identified, i.e., yeast Prp5 RNA helicase,5 yeast methionine aminoacyl tRNA synthetase,12 and human polyadenylate-binding protein 1 (PABPC1).200 Our findings led to the discovery of PFN1 as a reader protein for Ψ-containing RNA in mammalian cells. PFN1 is well-known for its role in actin polymerization, and TPI1 is a crucial glycolysis enzyme.36 The observed interaction between PFN1 and TPI1 mRNA suggests a potential link between actin cytoskeleton dynamics and cellular metabolism. It can be envisaged that PFN1 may help spatially coordinate metabolic processes with cytoskeletal rearrangement.

In summary, we profiled, for the first time, the Ψ-interaction proteome and uncovered a number of candidate Ψ-binding proteins, including cytoskeletal protein PFN1. We demonstrated that PFN1 interacts directly and selectively with Ψ-containing RNA, and that PFN1 and DKC1-mediated pseudouridylation regulates the stability and translation efficiency of a Ψ-containing mRNA (i.e., TPI1 mRNA) through modulating PFN1-Ψ interactions. Moreover, we uncovered other candidate Ψ-binding proteins, for which further characterizations are necessary for improving our understanding of the biological functions of Ψ and those RNA-binding proteins.

Acknowledgments

The authors thank the National Institutes of Health for supporting this research (R35 ES031707 to Y.W.). S.W. was supported in part by a T32 training grant (T32 ES018827).

Data Availability Statement

The PFN1 iCLIP data has been deposited into the NCBI GEO database with accession number GSE252793.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c17659.

  • Experimental procedures, data analysis, sequences for PCR primers, siRNAs, and the list of proteins quantified by the SILAC-based interaction screening (PDF)

The authors declare no competing financial interest.

Supplementary Material

ja4c17659_si_001.pdf (2MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ja4c17659_si_001.pdf (2MB, pdf)

Data Availability Statement

The PFN1 iCLIP data has been deposited into the NCBI GEO database with accession number GSE252793.

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