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Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis

ABSTRACT: To investigate the ribosomal RNA subpopulation types in a mutant created by CRISPR-Cas9 editing of rDNA copies in Arabidopsis thaliana / Original Abstract: Arabidopsis contains hundreds of ribosomal DNA copies organised within the nucleolar organising regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1-4, based on the polymorphisms of 3' external transcribed sequences. The variants are differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~25 nt from only the copies of NOR4 ribosomal DNA, obtaining mosaic mutational events on ~5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies, marked by its associated single-nucleotide variants and, thus, resulted in an altered rRNA sub-population. Furthermore, it caused rRNA maturation defects specifically in the minor pathway characterised by 32S pre-rRNA accumulation. The mutated rRNAs were not incorporated into the translating ribosomes, while it induced an elevated autophagic flux, likely related to ribophagy.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE213764 | GEO | 2024/01/10

REPOSITORIES: GEO

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Project description:Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and exclude a significant role for termination by a torpedomechanism. They further reveal the existence of an asymmetric Boundary Complex formed by CTCF, Cohesin and three phased nucleosomes lying adjacent to the rDNA Enhancer and an arrested RNA Polymerase I complex. We find that this complex is the only site of active histone modification in the whole 45kbp rDNA repeat. Strikingly, the Enhancer Boundary Complex not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define the poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for the chromatin remodelling complexes.

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Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis

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