RESUMO
RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Íntrons , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , RNA de Cadeia Dupla , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Interferons/metabolismo , Interferons/genética , Animais , Células HEK293 , Camundongos , Transcriptoma , Éxons , Sítios de Splice de RNA , Elementos Alu/genéticaRESUMO
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.
Assuntos
Proteína Centromérica A/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Proteína Centromérica A/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila , Proteínas de Drosophila/genética , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Mitose/genética , Mitose/fisiologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genéticaRESUMO
Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 "placeholder" nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. Here, we used Drosophila melanogaster tissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, we found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.