Anti-RPF2 monoclonal antibody, clone FQ21363

Rpf2 (Ribosome Production Factor 2) is involved in ribosome biogenesis, and functions as one of the pre-rRNA processing factors. Although properties of the Rpf2 protein in yeast have been described in detail, there have been no reports on Rpf2 homologues in higher eukaryotes. To investigate the function of Rpf2 homologues in higher plants, we identified genes encoding Rpf2-like proteins from Arabidopsis and rice and designated them AtRpf2 and OsRpf2, respectively. The predicted amino acid sequence revealed that both genes contain the Brix domain, a characteristic domain associated with ribosome biogenesis, and a sigma 70-like RNA binding motif, a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis. Both gene transcripts were detected in all organs and tissues tested and the expression patterns were consistent with the typical expression pattern of previously described ribosomal protein genes. Transient expression of GFP::AtRpf2 and GFP::OsRpf2 revealed predominant nucleolar localization within the nucleus of both fusion proteins in onion epidermal cells and tobacco BY-2 cells. These results suggest that Rpf2-like proteins in higher plants are also involved in ribosome biogenesis and function as pre-rRNA processing factors.

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p, is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.