SRSF1 as a novel interacting partner for IFITM1/3 unravels the emergent role of IFITM1/3 mediating protein expression

IFITM proteins play a role in cancer cell progression through undefined mechanisms. Here, we propose an emergent role of IFITM1/3 regulating protein synthesis. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the cytosolic isoform of SRSF1 that transports mRNA to the ribosome. This cytosolic association was confirmed in situ using proximity ligation assays for SRSF1 and IFITM1/3, suggesting a role associated with translation. Accordingly, IFITM1/3 were shown to interact with HLA-B mRNA in response to IFNγ stimulation using RNA-protein proximity ligation assays. In addition, shotgun RNA sequencing in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that reduced HLA-B gene expression does not account for lowered HLA-B protein synthesis in response to IFNγ. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to their wild-type counterpart, partially reverted by IFITM1/3 complementation. Our data all together link the binding of IFITM1/3 proteins to HLA-B mRNA and SRSF1 as a mechanism to catalyze the synthesis of target proteins, suggesting an RNA chaperonin role for IFITM1/3 proteins. Significance IFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. However, mechanistic insights are not well understood. Our study proposes that IFITMs have a role regulating protein synthesis, a pivotal function highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 is present in the ribosomal fraction and regulates particular protein expression; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs mediating protein translation is relevant, as has the potential of regulating the expression of viral and oncogenic proteins.


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white represent a low level of expression becoming non-significant. Finally, the values in grey are for 258 the genes in which TPM was equal to 0.

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Sucrose gradient sedimentation 261 For experiments processing ribosomal fractions using the wt-SiHa cells and IFITM1/IFITM3 262 null cells (Fig 5), the cultures were stimulated with 100 ng/ml IFNγ for 24 h prior to cell harvesting.

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In the complementation assays (SI Appendix, Fig. S5D  for 10 min and centrifuged at 10,000 g for 10 min at 4ºC. Lysates were processed as described (Sanford 273 et al., 2004). Sucrose gradients (10-45%) were prepared using a BioComp gradient master. Lysates 274 were applied onto the gradient and centrifuged at 41,000 rpm for 2 h 30 min using a SW41 rotor.

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Roche/Sigma-Aldrich). In addition, sample preparation for western blotting required 282 deproteinization of crude samples and was performed using the trichloroacetic acid (TCA) method.

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Cloning, transfection and affinity purification of IFITM1 from heavy isotope labeled cells 285 IFITM1 cDNA was cloned by PCR into pEXPR-IBA105 expression vector containing a SBP 286 tag at the N-terminus of the coding region (SBP vector, IBA, Germany). Cells were grown as biological 287 triplicates for 10 days with 5 passages in RPMI SILAC media before transfection (Dundee Cell

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For transfection, cells were grown to approximately 80% confluency in light and heavy media and 291 transfected using Attractene (#301007, Qiagen, Germany) with SBP-empty vector (control cells) and 292 SBP-IFITM1 (SI Appendix, Fig. S2). At 24 and 48 h after transfection, cells were washed twice in ice 293 cold PBS and scraped into 0.1% Triton buffer for 30 min on ice. Equal amounts of protein were used 294 for performing the pull down. Total protein extracts were measured by Bradford assay. For affinity 295 purification, the cells were washed twice in cold PBS and scraped directly into IP buffer (100 mM 296 KCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 10 mM NaF, 10% (v/v) 8 of 33 were eluted from the beads using Elution Buffer (20 mM HEPES pH 8, 2 mM DTT, and 8 M Urea).

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The eluted samples (whole volume) from light and heavy SILAC medium labeled cells were mixed 302 together.

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Peptide generation using FASP 305 Proteins eluted after SBP pull-down were processed by the filter-aided sample preparation

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Tryptic peptides from isotopically labeled cells were separated using an UltiMate 3000

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Database searching and analysis

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Data availability

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The mass spectrometry proteomics data have been deposited to the ProteomeXchange

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Thus, the endogenous IFITM1 and SRSF1 co-association was further validated by proximity ligation 389 assay (PLA), which can detect two endogenous protein-protein interactions in situ (Weibrecht et al., protein expression does not decrease these transcripts (Fig 3B IV;highlighted in red). In addition to 472 these classic IFNγ-inducible genes, other highly-induced transcripts after IFNγ treatment are also 473 revealed to be IFITM1/IFITM3-independent; these include HLA-DRB1, HLA-DRA, CXCL10 and 474 GBP5 (Fig 3 I and IV, highlighted in black). These genes are related to tumor immunity or pathogen 475 restriction (Mach et al., 1996;Soejima and Rollins, 2001;Feng et al., 2017). Nonetheless, globally, no 476 major differences were observed in the total amount of transcripts in the cells lacking of IFITM1/3 477 expression compared to wt-SiHa cells (Fig 3B).

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Furthermore, we focused on the mRNA abundance of the 31 genes composing the IRDS 483 cluster. The HLA-B is an IRDS gene whose expression seems to be mediated by IFITM1/3; therefore, 484 we investigated whether other transcripts originated from the IRDS genes may be affected. IRDS 485 genes derived from Table S2 are a subset of IFN responsive genes linked to chemo-radiation 486 resistance that include IFITM1 (but not IFITM3) and HLA-B (Erdal et al., 2017). In this regard, a heat

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In the attempt to study the relation of IFITM1/3 with HLA-B protein translation, we first 500 asked whether IFITM1/3 proteins are present in the ribosome by studying their potential interaction 501 with RPL7a protein, a ribosome-specific protein used as a subcellular marker. RPL7a is a component  integrity. Then, cell lysates from IFNγ-stimulated cells were subjected to sucrose gradient 515 sedimentation under conditions that maintain ribosome integrity (Fig 5). Immunoblots of the 516 polysomal fractions indicated that IFITM1/3 proteins can be detected in the 40S, 60S, and 80S fractions 517 from wt-SiHa cells but not from the IFITM1/IFITM3 null cells (Fig 5B).

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The representative A254 nm peaks of 40S, 60S, 80S and polysomal fractions is depicted in Fig   519   5A. Interestingly, the A254 absorbance scan of the 80S subunit fraction in the IFITM1/IFITM3 null 520 cells was reproducibly lower than in wt-SiHa cells in three independent biological replicates ( Fig 5A,

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To begin with, we start tackling this question by identifying new interacting partners for 550 IFITM that could shed some light on which novel role IFITM1/3 proteins can be implicated. We 551 identified multiple members of the SR family of splicing factors (SRSF1, SRSF2, SRSF3, SRSF6 and 552 U2AF1) as novel candidates to associate with IFITM1 protein by employing affinity capture assay 553 coupled to pulse-SILAC mass spectrometry (SI Appendix, Fig. S2). A Proximity ligation assay regulate, at least partially, the expression of MHC class I proteins. Remarkably, several studies 601 revealed HLA expression deficiencies in lymph node metastases compared to primary tumor 602 (Cromme et al., 1994;Ferns et al., 2016) Table S1: List of proteins enriched in SBP-IFITM1 pull down, Table S2:    Table S1). (D) Identified SRSF peptides in SBP-IFITM1 protein enrichment after 990 affinity purification in isotopically labelled wt-SiHa cells. Heavy isotopically labelled tryptic peptides identified from the SBP-991 IFITM1 affinity enrichment are highlighted in red for the SRSF family of proteins (SRSF1, SRSF2, SRSF3, SRSF6 and U2AF1).

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Underlined unique peptides confirm that these proteins are SRSF isoforms reported to shuttle between the cytoplasm and nucleus. from the RNA extraction was further used to analyze transcripts of interest by RT-qPCR using the three biological replicates (Fig   1002   3). For this purpose, the RNA was reverse transcribed into cDNA and (V) sequentially quantified and compared between samples.