Biology subjects

Enganti, R.

Publications and source records attributed to Enganti, R..

3 recordsLinked to original sources

bioRxiv2023

Sequence and epigenetic landscapes of active and silenced nucleolus organizers in Arabidopsis

Arabidopsis thaliana has two ribosomal RNA gene loci, nucleolus organizer regions NOR2 and NOR4, whose complete sequences remain undefined. Ultra-long DNA sequences assembled using an unconventional approach yielded 5.5 and 3.9 Mbp sequences for NOR2 and NOR4 (in the reference strain, Col-0), revealing their distinct gene subtype compositions. RNA sequencing and identification of genes associated with flow-sorted nucleoli of wild-type or silencing-defective mutant plants shows that most of NOR4 is comprised of active genes whereas most, but not all, NOR2 genes are epigenetically silenced. Long intervals of low CG and CHG methylation overlap regions of gene activity and gene subtype homogenization. Collectively, the data reveal the genetic and epigenetic landscapes of the NORs and implicate transcription in rRNA gene concerted evolution. One Sentence SummaryNOR2 and NOR4 sequences fill genome gaps and enable megabase-scale analyses of rRNA gene regulation and concerted evolution.

genomics↗

bioRxiv2023

A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana, rescuing mutant defects from global translation and gene expression to photosynthesis and growth

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the Target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here we report data from Arabidopsis thaliana, which indicate that plants expressing only a largely phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6A (RPS6A) paralog of eS6 functionally rescued double mutations in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6A lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. It also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and the vast majority of gene expression defects that were measured by transcriptomics and proteomics. However, compared to plants rescued with a phospho-enabled version of eS6A, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects, with little or no evidence for gain-of-function defects. As expected, the phospho-deficient eS6A also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6B paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

plant biology↗

bioRxiv2023

Multimegabase-scale DNA hypermethylation and condensed chromatin correlate with chromosome-specific rRNA gene silencing in Arabidopsis

Arabidopsis thaliana has two nucleolus organizer regions (NORs), consisting of long tandem arrays of 45S rRNA genes on chromosomes 2 (NOR2) and 4 (NOR4). The rRNA gene subtypes mapping to NOR2 are mostly silenced during development, whereas most of those mapping to NOR4 are active. Cytosine methylation of promoters plays a significant role in rRNA gene silencing, but recent findings demonstrate that gene body methylation also play a key role in gene silencing. Previous studies have demonstrated that CG methylation plays a role in rRNA gene silencing. However, it remains to be determined whether Chromomethylase 2 (CMT2)-mediated, RdDM-independent CHH methylation plays any role in rRNA gene silencing. To find out the relative importance of cytosine methylation in each of the sequence contexts (CG, CHG, and CHH, where H=A, T, or C), we studied multiple DNA methylation-deficient Arabidopsis mutants. Primarily, we show that the mutants that displayed higher losses of CG or CHH methylation at rDNA loci, display higher degrees of NOR2 rRNA gene silencing disruption. Using whole genome bisulfite sequencing data, we show that the loss of RdDM-independent CMT2, not the loss of RdDM-dependent DRM2, erases most of the CHH methylation at rDNA loci, while retaining CG methylation levels comparable to those of wild-type Col-0, demonstrating the sufficiency of CHH methylation loss for releasing rRNA gene silencing. Analysis of Arabidopsis and tomato NOR sequences revealed that in 45S rRNA gene sequences, cytosines mostly occur in the CHH context, followed by CG and the lowest being in CHG contexts. The predominance of CHH sites is even more pronounced when the rRNA gene promoter and spacer promoter(s) are considered (0-7% CG, 14-25% CHG, and 79-80% CHH), suggesting a mechanism to explain the stronger disruption of NOR2 gene silencing in mutants that display higher losses of CHH methylation. Our data also reveal a role for gene body methylation in rRNA gene silencing. We show that NOR2 genes are relatively hypermethylated compared to NOR4, consistent with findings from recent studies. By contrast, human rRNA genes have more CG sites than CHH and CHG, correlating with the evolutionary loss of CMTs in mammals. CMTs are plant-specific, which primarily methylate CHG and CHH sites. Our data define a critical role for CMT2-mediated RdDM-independent CHH methylation, in combination with MET1-mediated CG methylation, in rRNA gene silencing, which is entirely mediated by CG methylation in mammals.

plant biology↗