RESUMEN
Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.
Asunto(s)
Histonas , ARN Polimerasa II , Humanos , Histonas/genética , Histonas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Empalme del ARN , Transcripción Genética , Cromatina/genética , Empalme AlternativoRESUMEN
Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Disautonomía Familiar/genética , Histona Desacetilasas/genética , Fosfatidilserinas/administración & dosificación , Tubulina (Proteína)/genética , Empalme Alternativo/genética , Animales , Transporte Axonal/genética , Axones/efectos de los fármacos , Modelos Animales de Enfermedad , Disautonomía Familiar/tratamiento farmacológico , Disautonomía Familiar/patología , Exones/genética , Ganglios Espinales/crecimiento & desarrollo , Ganglios Espinales/patología , Histona Desacetilasa 6 , Histona Desacetilasas/biosíntesis , Humanos , Ratones , Ratones Noqueados , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Factor de Crecimiento Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fosfatidilserinas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5' splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5' splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5' splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3' splice sites; potentially mediating the rapid splicing of long introns.
Asunto(s)
Intrones/genética , Sitios de Empalme de ARN/genética , Transcripción Genética , Emparejamiento Base/genética , Secuencia de Bases , Exones/genética , Células HEK293 , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , ARN Polimerasa II/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transcriptoma/genéticaRESUMEN
Familial Dysautonomia (FD) is an autosomal recessive congenital neuropathy that results from a point mutation at the 5' splice site of intron 20 in the IKBKAP gene. This mutation decreases production of the IKAP protein, and treatments that increase the level of the full-length IKBKAP transcript are likely to be of therapeutic value. We previously found that phosphatidylserine (PS), an FDA-approved food supplement, elevates IKAP levels in cells generated from FD patients. Here we demonstrate that combined treatment of cells generated from FD patients with PS and kinetin or PS and the histone deacetylase inhibitor trichostatin A (TSA) resulted in an additive elevation of IKAP compared to each drug alone. This indicates that the compounds influence different pathways. We also found that pridopidine enhances production of IKAP in cells generated from FD patients. Pridopidine has an additive effect on IKAP levels when used in combination with kinetin or TSA, but not with PS; suggesting that PS and pridopidine influence IKBKAP levels through the same mechanism. Indeed, we demonstrate that the effect of PS and pridopidine is through sigma-1 receptor-mediated activation of the BDNF signaling pathway. A combination treatment with any of these drugs with different mechanisms has potential to benefit FD patients.