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Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching.

ABSTRACT: Class switch recombination (CSR) changes antibody isotype by replacing C? constant exons with different constant exons located downstream on the immunoglobulin heavy (IgH) locus. During CSR, transcription through specific switch (S) regions and processing of non-coding germline transcripts (GLTs) are essential for the targeting of activation-induced cytidine deaminase (AID). While CSR to IgG1 is abolished in mice lacking an I?1 exon donor splice site (dss), many questions remain regarding the importance of I exon dss recognition in CSR. To further clarify the role of I exon dss in CSR, we first evaluated RNA polymerase II (RNA pol II) loading and chromatin accessibility in S regions after activation of mouse B cells lacking I?1 dss. We found that deletion of I?1 dss markedly reduced RNA pol II pausing and active chromatin marks in the S?1 region. We then challenged the post-transcriptional function of I exon dss in CSR by using antisense oligonucleotides (ASOs) masking I exon dss on GLTs. Treatment of stimulated B cells with an ASO targeting I?1 dss, in the acceptor S?1 region, or I? dss, in the donor S? region, did not decrease germline transcription but strongly inhibited constitutive splicing and CSR to IgG1. Supporting a global effect on CSR, we also observed that the targeting of I? dss reduced CSR to IgG3 and, to a lesser extent, IgG2b isotypes. Altogether, this study reveals that the recognition of I exon dss first supports RNA pol II pausing and the opening of chromatin in targeted S regions and that GLT splicing events using constitutive I exon dss appear mandatory for the later steps of CSR, most likely by guiding AID to S regions.

SUBMITTER: Marchalot A

PROVIDER: S-EPMC7233311 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching.

Marchalot Anne A Ashi Mohamad Omar MO Lambert Jean-Marie JM Carrion Claire C Lecardeur Sandrine S Srour Nivine N Delpy Laurent L Le Pennec Soazig S

Frontiers in immunology 20200508

Class switch recombination (CSR) changes antibody isotype by replacing Cμ constant exons with different constant exons located downstream on the immunoglobulin heavy (<i>IgH</i>) locus. During CSR, transcription through specific switch (S) regions and processing of non-coding germline transcripts (GLTs) are essential for the targeting of activation-induced cytidine deaminase (AID). While CSR to IgG1 is abolished in mice lacking an Iγ1 exon donor splice site (dss), many questions remain regarding ...[more]

PMID: 32477332

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Project description:The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For vif mRNA, intron 1 must be removed while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented. Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion in viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis, we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer-guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use of locked nucleic acids (LNAs) impaired Vif expression and viral replication.IMPORTANCE Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

Dataset Information

Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching.

Publications

Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching.

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