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 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.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.