RESUMO
The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Proteína Fosfatase 2 , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/ultraestrutura , Terminação da Transcrição Genética , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Ligação Proteica , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Subunidades Proteicas/metabolismo , Subunidades Proteicas/químicaRESUMO
X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.
Assuntos
Distúrbios Distônicos/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Transtornos Parkinsonianos/genética , Retroelementos/genética , Transcrição Gênica/genética , Adolescente , Adulto , Metilação de DNA/genética , Feminino , Histona Acetiltransferases/genética , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Adulto JovemRESUMO
X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative movement disorder, caused by a founder retrotransposon insertion in an intron of the TAF1 gene. This insertion contains a polymorphic hexanucleotide repeat (CCCTCT)n, the length of which inversely correlates with the age at disease onset (AAO) and other clinical parameters, aligning XDP with repeat expansion disorders. Nevertheless, many other pathogenic mechanisms are conceivably at play in XDP, indicating that in contrast to other repeat disorders, the (CCCTCT)n repeat may not be the actual (or only) disease cause. Here, we summarize and discuss genetic and molecular aspects of XDP, highlighting the role of the hexanucleotide repeat in age-related disease penetrance and expressivity.
