Neumann, S. A. | Search bioRxiv

bioRxiv2026

Dynamic 2'-O-Me of rRNA regulates ribosome translation in Arabidopsis nucleolin1 mutants and during seed-to-seedling transition

Across all kingdoms of life, 2'-O-ribose methylation (2'-O-Me) is the most abundant modification of ribosomal RNAs (rRNAs), which are central to ribosome assembly and function. The 2'-O-Me of specific rRNA sites is catalysed by C/D-box small nucleolar ribonucleoprotein (snoRNP) complexes. However, how and when the landscape of plant rRNA 2'-O-Me modifications is remodelled remains largely unexplored. To study the regulation of rRNA 2'-O-Me and its impact on ribosome translation, we characterised Arabidopsis NUCLEOLIN 1 (NUC1)-depleted plants, which exhibit reduced 2'-O-Me at dozens specific rRNA sites, and investigated the impact of 2'-O-Me in ribosome translation during plant development. We demonstrated that 2'-O-Me is dynamically regulated during seed-to-seedling transition. Our analysis identified differentially methylated rRNA sites, which are specifically located in structurally critical regions of the ribosome. Notably, this hypomethylation is uncoupled from the global expression, assembly, or subcellular localisation of C/D-box snoRNPs. We further observed that polysomes with low 2'-O-Me levels not only accumulate but also exhibit differential mRNA loading. These polysomes characterised by reduced 2'-O-Me at specific rRNA sites, influence multiples aspects of translation, including ribosomes stalling at the initiation, elongation and termination steps. Together, these findings advance our understanding of the dynamic regulation of rRNA 2'-O-methylation and its functional role in ribosome-mediated translation in plants.

plant biology↗

bioRxiv2020

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

Levels of the second messenger bis-3-5-cyclic di-guanosinemonophosphate (c-di-GMP) determine when Streptomyces initiate sporulation to survive under adverse conditions. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor {sigma}WhiG. However, functions of the development-specific c-di-GMP diguanylate cyclases (DGCs) CdgB and CdgC, and the phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Global transcriptome analysis of {Delta}cdgB, {Delta}cdgC, {Delta}rmdA and {Delta}rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20 % of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores. Altogether, this study provides a global view of the c-di-GMP-dependent genes that contribute to the hyphae-to-spores transition and sheds light on the shared and specific functions of the key enzymes involved in c-di-GMP metabolism in S. venezuelae. ImportanceStreptomyces are important producers of clinical antibiotics. The ability to synthesize these natural products is connected to their developmental biology, which includes a transition from filamentous cells to spores. The widespread bacterial second messenger c-di-GMP controls this complex switch and is a promising tool to improve antibiotic production. Here, we analyzed the enzymes that make and break c-di-GMP in S. venezuelae by studying the genome-wide transcriptional effects of the DGCs CdgB and CdgC and the PDEs RmdA and RmdB. We found that the c-di-GMP specified by these enzymes has a global regulatory role. However, despite shared enzymatic activities, the four c-di-GMP enzymes have specialized inputs into differentiation. Altogether, we demonstrate that altering c-di-GMP levels through the action of selected enzymes yields characteristically distinct transcriptional profiles; this can be an important consideration when modulating c-di-GMP for the purposes of natural product synthesis in Streptomyces.

microbiology↗