In eukaryotes, rRNA transcription and ribosome biogenesis occur in a subnuclear compartment called the nucleolus. In this subcompartment, RNA polymerase I transcribes an rRNA precursor (pre-rRNA) that harbors the 18S, 25S/28S, and 5.8S rRNAs and several noncoding internal and external transcribed spacers (ITS and ETS, respectively) (Fig. ). The pre-rRNA is chemically modified and cleaved by endo- and exonucleases to produce the mature rRNAs. This process has been most extensively characterized in the yeast Saccharomyces cerevisiae
(for detailed reviews, see references 36
). In this organism, the primary 35S pre-rRNA is cleaved at sites A0
, and A2
to yield the 20S and 27SA2
pre-rRNA intermediates. These cleavage steps are mediated by components of the ~80S small-subunit (SSU) processome/90S preribosomes (4
). The 20S pre-rRNA, packaged into 43S preribosomes, is exported to the cytoplasm, where it is dimethylated by Dim1 and processed at site D to form the mature 18S rRNA and thereby the 40S ribosomal subunit (SSU). The 27SA2
pre-rRNAs, part of 66S preribosomes, can be processed via two pathways leading to the synthesis of the 5.8S and 25S large-subunit (LSU) rRNAs (Fig. ). Finally, the 5S rRNA is independently transcribed as a precursor by RNA polymerase III (Fig. ). Many of the cleavage steps in pre-rRNA processing are believed to be endonucleolytic; thus far, however, the enzymes responsible for most of these cleavages have not been identified. Two well-studied examples are the RNase MRP snoRNP, which cleaves at site A3
, and Rnt1, an endonuclease responsible for cleavage of the 3′ ETS (24
). One possible candidate for the cleavage at site D in the 20S pre-rRNA is Nob1, a protein that contains a putative PIN domain, which shares structural homology with several exonucleases and flap endonucleases (2
). Consistent with a role as a nuclease, conserved residues within the PIN domain are shown by genetic studies to be essential for its function (8
). However, it remains unclear whether Nob1 has endonucleolytic activity.
FIG. 1. Schematic representation of the pre-rRNA-processing pathway in the yeast Saccharomyces cerevisiae. RNA polymerase I transcribes the 35S pre-rRNA, which contains the mature rRNA sequences (18S, 5.8S, and 25S), ITS, and ETS. The 5.85 rRNA has both short (more ...)
Large-scale proteomic and genomic studies have identified over 150 pre-rRNA-processing factors and numerous preribosomal complexes (11
). Peng et al. devised a genome-wide microarray approach to reveal yeast proteins involved in RNA metabolism (35
). They identified a large number of proteins required for ribosome biogenesis, including many that had not yet been studied. One of them, Fap7, is an essential nuclear and cytoplasmic protein previously identified in a genetic screen for factors required for activation of a reporter construct during oxidative stress (21
). Fap7 belongs to a novel family of P-loop kinases, designated AD-004/AF2001-type kinases of unknown function (21
). This family of kinases is related to ribonucleoside kinases and harbors Walker A, an hhh(DE)XH-type Walker B motif characteristic for NTPases (h represents any hydrophobic residue), and a conserved arginine or lysine that is predicted to play an important role in the binding of the α-phosphates of ribonucleosides. There is also considerable evidence suggesting a role in ribosome biogenesis for Fap7: it has been copurified with tandem affinity purification (TAP)-tagged Enp1 (Enp1-TAP) (14
), a protein predominantly associated with 43S preribosomes, and it has been shown to interact with the SSU processome protein Utp13 in a yeast two-hybrid assay (21
). Taken together, these results strongly suggest that Fap7 has a direct role in pre-rRNA processing.
In this work, we demonstrate that Fap7 is strictly required for cleavage at site D but not at any other processing steps, indicating that it has a role in the final cleavage step that generates the mature SSU. Strikingly, genetic depletion of Fap7 results in the accumulation of unusually high levels of 20S pre-rRNA in the cytoplasm. In contrast to other known nonribosomal proteins whose depletion causes accumulation of the 20S pre-rRNA, Fap7 is probably not a structural component of 43S preribosomes but most likely transiently interacts with these complexes by directly binding Rps14, placing Fap7 in close proximity to the D cleavage site. In agreement with its predicted NTPase activity, we show that conserved residues in the putative Walker A and Walker B motifs which are predicted to be involved in nucleotide binding are important for Fap7 function in vivo. Collectively, our results support a model in which processing of the 20S pre-rRNA at site D in the cytoplasm requires a transient interaction between Fap7 and Rps14 within the 43S preribosome.