RESUMEN
5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.
Asunto(s)
Proteínas de Neoplasias , Biosíntesis de Proteínas , Proteínas de Neoplasias/genética , Canadá , Metilación , ARN Mensajero/genética , ARN/metabolismoRESUMEN
Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.
Asunto(s)
Aneuploidia , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Replicación del ADN , Línea Celular , Centrómero/genética , Proteína A Centromérica/genética , Cromatina/genética , Cromosomas Humanos/genética , Humanos , Fase SRESUMEN
Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-AOFF/ON ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4+ T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Proteína A Centromérica/metabolismo , Proteína B del Centrómero/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Nucleosomas/genética , Linfocitos T CD4-Positivos/citología , Sistemas CRISPR-Cas , Ciclo Celular , Línea Celular Tumoral , Centrómero/genética , Segregación Cromosómica/genética , Biología Computacional , Epigénesis Genética , Marcación de Gen , Humanos , Hibridación Fluorescente in Situ , Nucleosomas/metabolismo , ARN Interferente PequeñoRESUMEN
The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Animales , Centrómero/metabolismo , Pollos , Ciclina B/metabolismo , Proteínas del Citoesqueleto/metabolismo , Femenino , Humanos , Mitosis , Mutación , Oocitos/metabolismo , Fosforilación , Unión Proteica , Proteínas Recombinantes/metabolismo , Estrellas de MarRESUMEN
Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Huso Acromático/metabolismo , Línea Celular , Centriolos/metabolismo , Centrómero/fisiología , Proteína A Centromérica/fisiología , Centrosoma/metabolismo , Centrosoma/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/fisiología , Microtúbulos/metabolismo , Mitosis/fisiología , Huso Acromático/fisiología , Polos del Huso/metabolismoRESUMEN
Cytosolic DNA activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity, and cancer. cGAS is considered to be a sequence-independent DNA sensor with limited access to nuclear DNA because of compartmentalization. However, the nuclear envelope is a dynamic barrier, and cGAS is present in the nucleus. Here, we identify determinants of nuclear cGAS localization and activation. We show that nuclear-localized cGAS synthesizes cGAMP and induces innate immune activation of dendritic cells, although cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knockin mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent on LINE elements. The non-enzymatic N-terminal domain of cGAS determines nucleo-cytoplasmic localization, enrichment on centromeres, and activation of nuclear-localized cGAS. These results reveal a preferential functional association of nuclear cGAS with centromeres.
Asunto(s)
Centrómero/enzimología , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Inmunidad Innata/genética , Nucleotidiltransferasas/metabolismo , Adulto , Animales , Línea Celular , Núcleo Celular/enzimología , ADN , ADN Satélite , Femenino , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Nucleotidiltransferasas/química , Dominios ProteicosRESUMEN
CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/fisiología , Edición Génica , Células HeLa , Humanos , FosforilaciónRESUMEN
Human centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation. Without the continuing presence of CENP-A, CENP-B binding to alphoid DNA sequences becomes essential to preserve anchoring of CENP-C and the kinetochore to each centromere. Thus, there is a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA sequences bound by CENP-B in the maintenance of human chromosome segregation.