SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus

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Highlights

  • SRSF1-3 promotes the nuclear accumulation of AID.

  • SRSF1-3 forms a protein complex with AID in an AID C-terminus-independent manner.

  • SRSF1-3 contributes to IgV hypermutation in an AID C-terminus-dependent manner.

Abstract

Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSF1-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSF1-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, overexpression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSF1-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C-terminus-dependent manner.

Introduction

Germinal center B cells express activation-induced cytidine deaminase (AID), which converts dC to dU on single-stranded (ss) DNAs to allow somatic hypermutation (SHM) on Ig variable region genes (IgV) and class switch recombination (CSR) on Ig constant region genes, during antibody affinity maturation [1], [2]. The expression and activity of AID are regulated through a multi-layered process, including regulation of subcellular localization (reviewed in Ref. [3]). AID is predominantly localized in the cytoplasm by nuclear exclusion and cytoplasmic retention. However, the molecular mechanisms that shuttle AID between the cytoplasm and the nucleus have not been fully elucidated.

The chicken B cell line DT40 constitutively expresses AID, and somatic hypermutation (SHM) and another IgV-diversifying process, gene conversion (GCV), both of which are initiated by a common intermediate, occur spontaneously in DT40 cells [4], [5]. We previously found an important role for a splice variant of the serine/arginine (SR)-rich protein splicing factor 1 (SRSF1), referred to here as SRSF1-3, in SHM and GCV [6]. An engineered DT40 cell line, DT40-ASF, in which the endogenous SRSF1 gene is disrupted and the human SRSF1 cDNA is expressed [7], was unable to induce SHM and GCV, whereas introduction of SRSF1-3 into the DT40-ASF cells restored these IgV diversification events [6]. The prototypical SR protein, SRSF1 (formerly ASF/SF2), is a nucleocytoplasmic shuttling protein, which is not only essential for RNA splicing but also has a variety of roles in RNA metabolism [8].

SRSF1-3 is an alternatively spliced product of SRSF1, which shares the N-terminal region with SRSF1 but contains an alternate C-terminus [9]. Since the alternate C-terminus is not conserved between species, and does not include any other characteristic protein motifs that indicate its function, the molecular role of SRSF1-3 in IgV diversification remains unknown. Recently, several transcription-coupled factors have been shown to associate with AID, thereby forming a protein complex that induces SHM and CSR (reviewed in Ref. [3]). It has also been reported that AID co-localizes with splicing factors in sub-nuclear domains where SRSF1 is predominantly enriched [10]. The increase in the number of reports connecting AID and SRSF1 prompted us to explore whether SRSF1-3, an isoform of SRSF1, contributes to the nuclear localization of AID. In this study, we investigated a role for SRSF1-3 in the subcellular localization of AID using fluorescently tagged fusion proteins. The results suggest that SRSF1-3 promotes accumulation of AID in the nucleus during IgV diversification.

Section snippets

Cell lines

Wild-type DT40 cells and 293T cells were obtained from the RIKEN Cell Bank. DT40-ASF cells [7], DT40-SW cells [11], and DT40-SWΔC cells [12] were described previously. DT40 cells were cultured as described previously [11]. 293T cells were cultured in DMEM (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and 50 μM 2-mercaptoethanol at 37 °C in 5% CO2

Construction of expression vectors and transfection

The FLAG-tagged chicken SRSF1-3 (FLAG-SRSF1-3) expression vector has been described previously [6]. An mCherry

Co-expression of AID with SRSF1-3 promotes the accumulation of AID in the nucleus

To visualize the subcellular localization of AID and SRSF1-3, chicken AID tagged with GFP at the C-terminus (AID-GFP) and chicken SRSF1-3 tagged with mCherry at the N-terminus (mCherry-SRSF1-3) were transiently expressed in human 293T cells. The subcellular localization of these fluorescent fusion proteins in these cells was then observed by confocal microscopy. When AID-GFP was expressed alone, i.e., without exogenous expression of SRSF1-3, AID-GFP was found predominantly in the cytoplasm in

Discussion

In the present study, overexpression of SRSF1-3 revealed that SRSF1-3 may induce nuclear accumulation of AID and promote IgV diversification in an AID C-terminus-dependent manner. SRSF1-3 formed a protein complex with AID, even in the absence of the AID C-terminus, as has been reported for other transcription-coupled factors that are responsible for SHM and CSR (reviewed in Ref. [3]), and furthermore SRSF1-3 and AID have been previously shown to be recruited to the IgV gene [6]. Thus, we

Acknowledgments

We are grateful to Prof. Hitoshi Ohmori for critical discussion and encouragement in the early phase of this work. We also acknowledge Dr. Ayano Satoh for providing helpful advice and support for microscopic analyses. We would like to thank Prof. James L. Manley for providing the DT40-ASF cells. This study was supported in part by JSPS KAKENHI Grants (26650126, 15H04196, 16K14783), Takeda Science Foundation, and Naito Foundation to N. Kanayama.

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    Although work carried out in the past three decades has identified multiple SRSF1 functions, and its pleiotropic effects on the immune system, the full regulatory potential of this protein is yet to be defined. For example, SRSF1−3, a splice isoform of SRSF1, is necessary for the somatic hypermutation (SHM) of Ig genes, which is initiated by the activation-induced cytidine deaminase (AID) [91,92]. Nevertheless, the exact molecular mechanism of action of SRSF1 in SHM remains unknown.

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    Interestingly, an important RNA splicing regulator SRSF1–3, a splicing isoform of SRSF1, has been reported to be required for AID-dependent SHM, as deletion of SRSF1–3 in DT40-ASF cells leads to depletion of SHM (Kanehiro et al., 2012). In addition, AID is reported to be co-localized with SRSF1–3 in the nucleus (Kawaguchi et al., 2017). Thus, SRSF1–3 is essential for SHM as well as the targeting of AID to Ig genes.

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    Since the SRSF1-3 deficient control cells do not contain FLAG-tagged SRSF1-3, the input samples do not show any band for SRSF1-3 (Fig. 5B, 5E panel 1). To confirm the earlier reports of interaction between SRSF1-3 and AID in human 293 T cells (Kawaguchi et al., 2017), we performed co-immunoprecipitation using the anti-FLAG antibody and detected AID by western blotting using the anti-AID antibody (Table S1), which shows a specific band at 24 kDa in SRSF1-3 reconstituted cells and is missing in the SRSF1-3 deficient cells (Fig. 5F and S4A). Subsequently, we confirmed this interaction by performing co-immunoprecipitation using the anti-AID antibody and detected SRSF1-3-FLAG bands using the anti-FLAG antibody (Table S1) in the reconstituted cells, as compared with the SRSF1-3 deficient cells (Fig. 5G and S4B), confirming an interaction of SRSF1-3 with AID, which probably influences the SHM of Ig genes.

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1

Present address: Department of Microbiology and Immunology, Shimane University School of Medicine, Izumo, Shimane 693-8501, Japan.

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