Overexpression of a Brix Domain-Containing Ribosome Biogenesis Factor ARPF2 and its Interactor ARRS1 Causes Morphological Changes and Lifespan Extension in Arabidopsis thaliana

The Brix domain is a conserved domain in several proteins involved in ribosome biogenesis in yeast and animals. In the Arabidopsis genome, six Brix domain-containing proteins are encoded; however, their molecular functions have not been fully characterized, as yet. Here we report the functional analysis of a Brix domain-containing protein, ARPF2, which is homologous to yeast Rpf2 that plays an essential role in ribosome biogenesis as a component of the 5S ribonucleoprotein particle. By phenotypic characterization of arpf2 mutants, histochemical GUS staining, and analysis using green fluorescence protein, we show that ARPF2 is an essential and ubiquitously expressed gene encoding a nucleolar protein. Co-immunoprecipitation and split-GFP-based bimolecular fluorescence complementation assays revealed that ARPF2 interacts with a protein named ARRS1, which is homologous to yeast Rrs1 that forms a complex with Rpf2 in yeast. Furthermore, the result of RNA immunoprecipitation assay indicated that ARPF2 interacts with 5S ribosomal RNA (rRNA) or the precursor of 5S rRNA, as well as with the internal transcribed spacer 2 in the precursors of 25S rRNA. Most intriguingly, we found that the overexpression of ARPF2 and ARRS1 leads to characteristic phenotypes, including short stem, abnormal leaf morphology, and long lifespan, in Arabidopsis. These results suggest that the function of Brix domain-containing ARPF2 protein in ribosome biogenesis is intimately associated with the growth and development in plants

Arabidopsis RPT2a, 19S Proteasome Subunit, Regulates Gene Silencing via DNA Methylation

The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation

Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system

The ubiquitin-proteasome system (UPS) is a unique protein degradation mechanism conserved in the eukaryotic cell. In addition to the control of protein quality, UPS regulates diverse cellular signal transduction via the fine-tuning of target protein degradation. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and response to biotic and abiotic stresses. Recent studies reveal that the UPS plays an essential role in adaptation to carbon and nitrogen availability in plants. Here we highlight ubiquitin ligase ATL31 and the homolog ATL6 target 14-3-3 proteins for ubiquitylation to be degraded, which control signaling for carbon and nitrogen metabolisms and C/N balance response. We also give an overview of the UPS function involved in carbon and nitrogen metabolisms

The bRPS6-Family Protein RFC3 Prevents Interference by the Splicing Factor CFM3b during Plastid rRNA Biogenesis in Arabidopsis thaliana

Plastid ribosome biogenesis is important for plant growth and development. REGULATOR OF FATTY ACID COMPOSITION3 (RFC3) is a member of the bacterial ribosomal protein S6 family and is important for lateral root development. rfc3-2 dramatically reduces the plastid rRNA level and produces lateral roots that lack stem cells. In this study, we isolated a suppressor of rfc three2 (sprt2) mutant that enabled recovery of most rfc3 mutant phenotypes, including abnormal primary and lateral root development and reduced plastid rRNA level. Northern blotting showed that immature and mature plastid rRNA levels were reduced, with the exception of an early 23S rRNA intermediate, in rfc3-2 mutants. These changes were recovered in rfc3-2 sprt2-1 mutants, but a second defect in the processing of 16S rRNA appeared in this line. The results suggest that rfc3 mutants may be defective in at least two steps of plastid rRNA processing, one of which is specifically affected by the sprt2-1 mutation. sprt2-1 mutants had a mutation in CRM FAMILY MEMBER 3b (CFM3b), which encodes a plastid-localized splicing factor. A bimolecular fluorescence complementation (BiFC) assay suggested that RFC3 and SPRT2/CFM3b interact with each other in plastids. These results suggest that RFC3 suppresses the nonspecific action of SPRT2/CFM3b and improves the accuracy of plastid rRNA processing