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

Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing 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 known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, 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 by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

Dynamics and thermal sensitivity of ribosomal RNA maturation paths in plants.

Ribosome biogenesis is a constitutive fundamental process for cellular function. Its rate of production depends on the rate of maturation of precursor ribosomal RNA (pre-rRNA). The rRNA maturation paths are marked by four dominant rate-limiting intermediates with cell-type variation of the processivity rate. We have identified that high temperature stress in plants, while halting the existing pre-rRNA maturation schemes, also transiently triggers an atypical pathway for 35S pre-rRNA processing. This pathway leads to production of an aberrant precursor rRNA, reminiscent of yeast 24S, encompassing 18S and 5.8S rRNA that do not normally co-occur together at sub-unit levels; this response is elicited specifically by high and not low temperatures. We show this response to be conserved in two other model crop plant species (Rice and Tomato). This pathway persists even after returning to normal growth conditions for 1 hour and is reset between 1-6 hours after stress treatment, likely, due to resumption of normal 35S pre-rRNA synthesis and processing. The heat-induced ITS2 cleavage-derived precursors and stalled P-A2-like precursors were heterogeneous in nature with a fraction containing polymeric (A) tails. Furthermore, high temperature treatment and subsequent fractionation resulted in polysome and precursor rRNA depletion.

The Arabidopsis 2'-O-Ribose-Methylation and Pseudouridylation Landscape of rRNA in Comparison to Human and Yeast.

Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2'-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.

The Existence and Localization of Nuclear snoRNAs in Arabidopsis thaliana Revisited.

Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification.

Plant-specific ribosome biogenesis factors in Arabidopsis thaliana with essential function in rRNA processing.

rRNA processing and assembly of ribosomal proteins during maturation of ribosomes involve many ribosome biogenesis factors (RBFs). Recent studies identified differences in the set of RBFs in humans and yeast, and the existence of plant-specific RBFs has been proposed as well. To identify such plant-specific RBFs, we characterized T-DNA insertion mutants of 15 Arabidopsis thaliana genes encoding nuclear proteins with nucleotide binding properties that are not orthologues to yeast or human RBFs. Mutants of nine genes show an altered rRNA processing ranging from inhibition of initial 35S pre-rRNA cleavage to final maturation events like the 6S pre-rRNA processing. These phenotypes led to their annotation as 'involved in rRNA processing' - IRP. The irp mutants are either lethal or show developmental and stress related phenotypes. We identified IRPs for maturation of the plant-specific precursor 5'-5.8S and one affecting the pathway with ITS2 first cleavage of the 35S pre-rRNA transcript. Moreover, we realized that 5'-5.8S processing is essential, while a mutant causing 6S accumulation shows only a weak phenotype. Thus, we demonstrate the importance of the maturation of the plant-specific precursor 5'-5.8S for plant development as well as the occurrence of an ITS2 first cleavage pathway in fast dividing tissues.

Late ribosomal protein localization in Arabidopsis thaliana differs to that in Saccharomyces cerevisiae.

Ribosome biogenesis is essential for cellular function and involves rRNA synthesis, rRNA processing and modification, and ribosomal protein assembly. Ribosome biogenesis factors and small nucleolar RNA assist these events. Ribosomal maturation takes place in the nucleolus, the nucleoplasm, and the cytosol in a coordinated and controlled manner. For example, some ribosomal proteins are thought to be assembled in the cytoplasm based on the observations in Saccharomyces cerevisiae. Here, we used cellular fractionation to demonstrate that cleavage of the 20S intermediate, the precursor to mature 18S rRNA, does not occur in the nucleoplasm of Arabidopsis thaliana. It most likely occurs in the cytoplasm. Further, we verified the proposed localization of RPS10e, RPS26e, and RPL24a/b in the nucleus and RPP1 in the nucleolus of A. thaliana by ribosome profiling, immunofluorescence, and analysis of the localization of GFP fusion proteins. Our results suggest that the order of events during ribosomal protein assembly in the ribosome biogenesis pathway differs between plants and yeast.