No abstract

Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants.

SUMMARYRibosome biogenesis involves a large ensemble of trans-acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre-60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C-terminus, which is highly charged in atLSG1-2 but missing in atLSG1-1. This C-terminus of atLSG1-2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1-2 to the nucleolus. Furthermore, only atLSG1-2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T-DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1-2 18S rRNA precursors accumulate and a 20S pre-rRNA is detected, while the amount of pre-rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre-60S subunit maturation is important for the final steps of pre-40S maturation in plants. In addition, the lsg1-2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.

Ribosome biogenesis is an essential process in all organisms. In eukaryotes, multiple ribosome biogenesis factors (RBFs) act in the processing of ribosomal (r)RNAs, assembly of ribosomal subunits and their export to the cytoplasm. We characterized two genes in Arabidopsis thaliana coding for orthologs of yeast BRX1, a protein involved in maturation of the large ribosomal subunit. Both atBRX1 proteins, encoded by AT3G15460 and AT1G52930, respectively, are mainly localized in the nucleolus and are ubiquitously expressed throughout plant development and in various tissues. Mutant plant lines for both factors show a delay in development and pointed leaves can be observed in the brx1-2 mutant, implying a link between ribosome biogenesis and plant development. In addition, the pre-rRNA processing is affected in both mutants. Analysis of the pre-rRNA intermediates revealed that early processing steps can occur either in the 5 ′ external transcribed spacer (ETS) or internal transcribed spacer 1 (ITS1). Interestingly, we also find that in xrn2 mutants, early processing events can be bypassed and removal of the 5 ′ ETS is initiated by cleavage at the P ′ processing site. While the pathways of pre-rRNA processing are comparable to those of yeast and mammalian cells, the balance between the two processing pathways is different in plants. Furthermore, plant-specific steps such as an additional processing site in the 5 ′ ETS, likely post-transcriptional processing of the early cleavage sites and accumulation of a 5 ′ extended 5.8S rRNA not observed in other eukaryotes can be detected.