atBRX1-1 and atBRX1-2 are involved in an alternative rRNA processing pathway in Arabidopsis thaliana

Abstract

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.

Plant-Specific Features of Ribosome Biogenesis

“…Pre-rRNA processing in A. thaliana, and likely also in other plant species, follows at least two alternative routes ( Figure 1, main text), and these appear to be a mixture between the mammalian and yeast pre-rRNA processing pathways [49,51]. However, these alternative routes are more independent of each other compared with mammalian cells and one independent processing site has also been identified [42,49].…”

“…However, these alternative routes are more independent of each other compared with mammalian cells and one independent processing site has also been identified [42,49]. The coexistence of alternative routes is thought to secure the generation of sufficient amounts of ribosomes or gives rise to regulation under certain stress situations [49][50][51].…”

“…However, these alternative routes are more independent of each other compared with mammalian cells and one independent processing site has also been identified [42,49]. The coexistence of alternative routes is thought to secure the generation of sufficient amounts of ribosomes or gives rise to regulation under certain stress situations [49][50][51]. Consistent with this notion, the analysis of RBFs in A. thaliana and other plant species revealed a tight connection between ribosome biogenesis and development of plant organs such as cotyledons and young leaves [38,59,75,82]; in addition, and many genes encoding plant RPs are under the control of TOR, a master regulator of cell cycle control [69,70].…”

“…The resulting prerRNA still contains the complete 5 0 -ETS, including the A 123 B cluster and the plant-specific 1083-nucleotide (nt) insertion of unknown function. After a speculated splicing event, XRN2 and the plant U3 snoRNP are involved in P cleavage, leading to the 35S pre-rRNA [33,34,42,49], which is then processed in two alternative ways, simultaneously with the 3 0 -ETS removal. In pathway 1 (5 0 -ETS-first) the 5 0 -ETS is removed through subsequent cleavages at sites P' and P 2 , leading to 33S(P 0 ) and 32S pre-rRNAs.…”

“…Barely detectable prerRNAs (33SP 0 , P 1 -A 3 and 20S) are shown in gray text. Modified from [49] (B). Abbreviations: ETS, external transcribed spacer; IGS, intergenic spacer; ITS, internal transcribed spacer.…”