RESUMEN
In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.
Asunto(s)
Arabidopsis/genética , Cromatina/metabolismo , ADN Ribosómico/genética , Duplicación de Gen , Fosfoproteínas/genética , ARN Ribosómico/genética , Proteínas de Unión al ARN/genética , Variaciones en el Número de Copia de ADN , Metilación de ADN , Genes de Plantas , Regiones Promotoras Genéticas , NucleolinaRESUMEN
In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Genes de ARNr/genética , Mutación/genética , Fosfoproteínas/metabolismo , ARN de Planta/genética , Proteínas de Unión al ARN/metabolismo , Arabidopsis/enzimología , ADN Espaciador Ribosómico/genética , Perfilación de la Expresión Génica , Histonas/metabolismo , Región Organizadora del Nucléolo/genética , Nucleosomas/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , ARN Polimerasa I/metabolismo , ARN Ribosómico/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transcripción Genética , NucleolinaRESUMEN
While plant rRNA gene organization and expression have been studied for several decades, the repetitive nature and high sequence identity between the tandemly-repeated units has prevented precise studies to determine which units are active and elucidate mechanisms of spatial and temporal transcriptional control. We have detected four variants among rRNA genes of Arabidopsis thaliana by analysis of the 3' external spacer region. Surprisingly, the most abundant variant, representing ~50% of the genes, is not expressed in wild-type plants but is transcribed in lines mutated in one of the two nucleolin genes. Analysis of Arabidopsis ecotypes indicated considerable variability in numbers and presence/absence of variants, although more closely-related ecotypes show similar profiles. Sequence analysis showed that one of the variants is not only unexpectedly located within a 5S RNA gene cluster in the pericentromeric region of chromosome 3 but is also potentially highly expressed in wild-type plants. We present a model to explain how transcription of this this unusual variant, maintained in an active state by nucleolin binding, could be involved in control of expression of the major variant, while absence of nucleolin leads to silencing of the minor variant and consequent expression of the major variant.