The Function of RH22, a DEAD RNA Helicase, in the Biogenesis of the 50S Ribosomal Subunits of Arabidopsis Chloroplasts    

Abstract

The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.Given the prokaryotic origin of chloroplasts, the protein-synthesizing systems of chloroplasts are often considered to be very similar to those of bacteria. Chloroplasts contain 70S ribosomes, which are similar to prokaryotic ribosomes and distinct from their cytosolic counterparts, 80S ribosomes. The ribosomal RNAs (rRNAs) of higher plant chloroplasts are highly conserved and are most closely related to bacterial sequences (Branlant et al., 1981;Harris et al., 1994). The 30S subunit comprises 16S rRNA in chloroplasts and Escherichia coli. However, the 50S subunit of chloroplast ribosomes is composed of three rRNAs (23S, 4.5S, and 5S rRNAs), rather than the 23S and 5S rRNAs of E. coli (Harris et al., 1994). In chloroplasts, the 16S, 23S, 4.5S, and 5S rRNAs are encoded in an operon and are synthesized as a single large precursor (Edwards and Kö ssel, 1981

RHON1 Mediates a Rho-Like Activity for Transcription Termination in Plastids of Arabidopsis thaliana

“…Probes were generated by PCR amplification of the relevant chloroplast sequences and labeled by the random priming method. The ribonuclease-protection assay of the RNA samples was performed as described by Chi et al (2012).…”

Chloroplast RH3 DEAD Box RNA Helicases in Maize and Arabidopsis Function in Splicing of Specific Group II Introns and Affect Chloroplast Ribosome Biogenesis

“…Arabidopsis mutant nara12-1, with reduced accumulation of RH39, showed strongly reduced levels of the photosynthesis machinery due to inefficient translation elongation, most likely due to the loss of RH39-mediated defects in 23S rRNA maturation (formation of the hidden break) affecting the functional state of the ribosome (Nishimura et al, 2010). Complete loss of Arabidopsis RH22 resulted into embryo lethality, whereas reduced RH22 expression resulted in virescent seedlings in which precursors of 23S and 4.5S rRNA (in the 50S particle) accumulated (Chi et al, 2012). It was concluded that RH22 facilitates the assembly of the 50S ribosomal subunit by interacting with ribosomal protein RPL24 and with the region of 23S rRNA encompassing the RPL24 binding site (Chi et al, 2012).…”

“…Complete loss of Arabidopsis RH22 resulted into embryo lethality, whereas reduced RH22 expression resulted in virescent seedlings in which precursors of 23S and 4.5S rRNA (in the 50S particle) accumulated (Chi et al, 2012). It was concluded that RH22 facilitates the assembly of the 50S ribosomal subunit by interacting with ribosomal protein RPL24 and with the region of 23S rRNA encompassing the RPL24 binding site (Chi et al, 2012). Tobacco mutants with loss of the RH58 homolog (VDL helicase) showed pale-green and variegated leaves and abnormal roots and flowers, but no function was determined (Wang et al, 2000).…”

DEAD-box RNA helicases and epigenetic control of abiotic stress-responsive gene expression

“…In plants, DEADbox RNA helicases have been associated with diverse molecular functions such as siRNA-mediated gene silencing, premRNA splicing, chloroplast/mitochondrial intron splicing, nonsense-mediated mRNA decay, mRNA export, ribosome biogenesis and exon-junction complex functions. [11][12][13][14][15][16][17][18][19][20][21][22] Furthermore, it has been shown that these helicases play roles in Keywords: abiotic stress, Arabidopsis thaliana, DEAD-box RNA helicases, epigenetic gene silencing, nucleolus, RNAdirected DNA methylation Abbreviations: ABA, abscisic acid; STRS, STRESS RESPONSE SUPPRESSOR; RdDM, RNA-directed DNA methylation. various biological processes including abiotic stress responses.…”