Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

Abstract

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.gene conversion | genomic instability S omatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes are key processes needed to generate functional antibodies (Abs) with high affinity in humoral immune responses. Activation-induced cytidine deaminase (AID) is crucial for both SHM and CSR (1, 2), which are initiated by AIDcatalyzed deamination of dC to dU in the variable (V) region and the switch (S) region DNA strands, respectively (3, 4). However, AID has been recently shown to bind numerous chromosomal locations genome-wide and to mutate non-Ig genes, although the mutation frequency of non-Ig genes is much lower than that of the Ig genes (5, 6). It has been proposed that specific cofactors interact with AID and regulate its activity in a target-specific manner (7). AID has been reported to associate with several proteins including RPA (8, 9), protein kinase A (10), RNA polymerase II (Pol II) (11), DNA-PKcs (12), MDM2 (13), Spt6 (14), 14-3-3 (15), Spt5 (16), CTNNBL1 (17), PTBP2 (18), RNA exosome subunits (19), KAP1 (20), and HP1 (20). However, these investigations were mostly focused on CSR, and, thus, the identified factors themselves are not sufficient to explain how AID activity is specifically targeted to the IgV genes during SHM.SHM occurs in IgV region exons starting 100-200 bp downstream of the promoter and extending over 1-2 kb, preferentially targeting WRC/GYW hotspot motifs (3, 4). Importantly, the activity of the SHM machinery strictly depends on transcription of the IgV genes (21, 22). In vitro experiments have shown that replication protein A (RPA) binds to and stabilize...

A Novel Regulator of Activation-Induced Cytidine Deaminase/APOBECs in Immunity and Cancer: Schrödinger’s CATalytic Pocket

“…To date, almost all efforts have focused on modes of regulation that are extrinsic to the enzyme itself. This has led to the identification of over two dozen co-factors proposed to bind AID either directly or indirectly through associations with other proteins or DNA/RNA (75–99). The list of putative binding partners is rather large for a relatively small protein of 198 residues.…”

T Cell Activation Regulates CD6 Alternative Splicing by Transcription Dynamics and SRSF1

“…SRSF1 is a prototype member of the serine/arginine-rich protein family of splicing factors with a role in alternative splicing (58)(59)(60)(61). SRSF1 has been extensively studied; however, only a few endogenous target genes have been identified (62)(63)(64)(65). Our results indicate that, upon T cell activation, the deficit of SRSF1 may be crucial for exon 5 exclusion, with SRSF1 most likely becoming a limiting factor for the increased levels of CD6 transcription and CD6Dd3 mRNA increase detected upon T cell activation.…”

Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers

“…Meanwhile, an Igλ regulatory element (Region A) was found to play a role in recruiting AID to the Igλ locus and is therefore important for AID-mediated GCV in chicken B cells (90,91). A splice isoform of the prototypical serine/arginine-rich (SR) protein splicing factor SRSF1 (SRSF1-3) binds preferentially to the IgV gene and makes it available for AID-induced SHM in a DT40 chicken B cell line (92). …”