Abstract
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.
Highlights
Ribosome biogenesis is a complex process that requires numerous factors, including ribosomal proteins (RPs), ribosomal RNAs, and ribosome biogenesis factors (RBFs) (Henras et al, 2015; Weis et al, 2015a)
Brix Domain-Containing Arabidopsis ARPF2 the maturation steps, the 45S pre-ribosomal RNA (rRNA) in the 90S pre-ribosomal particle is processed into 18S, 5.8S, and 25S rRNA by the elimination of 5 - and 3 -external spacer regions [5 -external transcribed spacer (ETS) and 3 -ETS] and two internal spacer regions [internal transcribed spacer (ITS) 1 and ITS2], and the 90S pre-ribosomal particle is divided into the 40S preribosome subunit, containing 18S rRNA, and the 60S preribosome subunit, containing 5.8S and 25S rRNAs (Henras et al, 2015; Weis et al, 2015a)
We investigated whether ARPF2 got bound to pre-rRNA, as yeast Rpf2 was shown to interact with 5S rRNA directly and with 25S rRNA directly or indirectly (Asano et al, 2015; Kharde et al, 2015; Madru et al, 2015)
Summary
Ribosome biogenesis is a complex process that requires numerous factors, including ribosomal proteins (RPs), ribosomal RNAs (rRNAs), and ribosome biogenesis factors (RBFs) (Henras et al, 2015; Weis et al, 2015a). Rpf alone can bind to 5S rRNA, Rpf was shown to more rigidly bind to 5S rRNA through forming the complex with Rrs (Madru et al, 2015) Another Brix domain-containing protein, Imp, was found to bind to U3 small nucleolar RNA and a U3 small nucleolar RNA-associated protein, Mpp, through its Brix domain and it played a role in the nucleolar processing of pre-18S ribosomal RNA (Lee and Baserga, 1999; Granneman et al, 2003). The Brix domain is supposed as a structural hub for interactions with both protein and RNA that are mediated by its N-terminal and C-terminal halves, respectively
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