Physiological and druggable skipping of immunoglobulin variable exons in plasma cells

Abstract

The error-prone V(D)J recombination process generates considerable amounts of nonproductive immunoglobulin (Ig) pre-mRNAs. We recently demonstrated that aberrant Ig chains lacking variable (V) domains can be produced after nonsense-associated altered splicing (NAS) events. Remarkably, the expression of these truncated Ig polypeptides heightens endoplasmic reticulum stress and shortens plasma cell (PC) lifespan. Many questions remain regarding the molecular mechanisms underlying this new truncated Ig exclusion (TIE-) checkpoint and its restriction to the ultimate stage of B-cell differentiation. To address these issues, we evaluated the extent of NAS of Ig pre-mRNAs using an Ig heavy chain (IgH) knock-in model that allows for uncoupling of V exon skipping from TIE-induced apoptosis. We found high levels of V exon skipping in PCs compared with B cells, and this skipping was correlated with a biallelic boost in IgH transcription during PC differentiation. Chromatin analysis further revealed that the skipped V exon turned into a pseudo-intron. Finally, we showed that hypertranscription of Ig genes facilitated V exon skipping upon passive administration of splice-switching antisense oligonucleotides (ASOs). Thus, V exon skipping is coupled to transcription and increases as PC differentiation proceeds, likely explaining the late occurrence of the TIE-checkpoint and opening new avenues for ASO-mediated strategies in PC disorders.

New models to study plasma cells in mouse based on the restriction of IgJ expression to antibody secreting cells

“…We now showed that CH1 deletion directly occurring in PCs is highly toxic, as previously shown in our laboratory for truncated LCs 30 , paving the way for new therapeutics targeting monoclonal Ig. Currently, studies using of Antisense Oligo Nucleotides (ASOs) to induce exon skipping of Ig genes are being developed and could be adapted to be used in PC malignancies to generate toxic truncated Igs 42 . Altogether, we herein described two new models to study PCs using the restricted expression of IgJ gene to antibody secreting cells.…”

Uncoupling splicing from transcription using antisense oligonucleotides reveals a dual role for I exon donor splice sites in antibody class switching

“…Consequently, using classical I exon deletion and/or replacement mouse models neither allow distinguishing the roles of I exon dss in the interconnected processes of transcription and splicing nor permit distinguishing between requirements of splicing per se from that of stable S GLTs 36 . Several studies including ours have shown the efficacy of ASO-mediated approaches to modify RNA splicing in B-lineage cells 26,37,38 . Here, we used ASOs targeting short sequence (23 to 25 nucleotides) spanning the Iγ1 or Iμ exon dss on pre-mRNA to analyse the consequences of GLTs splicing defect on CSR to IgG1 in splenic B cells from WT mice.…”

“…For this purpose, we treated stimulated splenic B cells isolated from WT mice with vivo-morpholino antisense oligonucleotide targeting the dss sequence of Iγ1 exon (Iγ1 dss ASO) (Figure 2A). Indeed, we previously demonstrated that passive administration of these Phosphorodiamidate Morpholino Oligonucleotides complexed with an octaguanidine dendrimer could very efficiently modulate the splicing of Ig transcripts 26 . First, we wanted to verify that masking the dss sequence of Iγ1 exon on GLTs by ASO did not inhibit γ1 GL transcription.…”

Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphocytes

“…Our laboratory recently examined the magnitude of NAS during B-cell development and PC differentiation in vivo using the previously mentioned IgH frVκ/+ mice harboring an additional PTC-containing V exon on one IgH allele [111]. This model facilitates the quantification of NAS because Ig heavy chains can be produced after skipping of the PTC-containing extra-V exon.…”

“…Interestingly, NAS of PTC-containing IgH RNAs was much more pronounced in PCs compared to B cells. The analysis of IgH transcription in different B and PC populations also revealed that the boost of Ig gene transcription accompanying PC differentiation correlated with high levels of NAS [111]. On one hand, alternative splicing is closely correlated to the rate of RNA polymerase II elongation and exon skipping is preferentially observed for highly transcribed genes [112,113].…”