Abstract
The AAA + ATPase R2TP complex facilitates assembly of a number of ribonucleoprotein particles (RNPs). Although the architecture of R2TP is known, its molecular basis for acting upon multiple RNPs remains unknown. In yeast, the core subunit of the box C/D small nucleolar RNPs, Nop58p, is the target for R2TP function. In the recently observed U3 box C/D snoRNP as part of the 90 S small subunit processome, the unfolded regions of Nop58p are observed to form extensive interactions, suggesting a possible role of R2TP in stabilizing the unfolded region of Nop58p prior to its assembly. Here, we analyze the interaction between R2TP and a Maltose Binding Protein (MBP)-fused Nop58p by biophysical and yeast genetics methods. We present evidence that R2TP interacts largely with the unfolded termini of Nop58p. Our results suggest a general mechanism for R2TP to impart specificity by recognizing unfolded regions in its clients.
Highlights
Box C/D type of small nucleolar ribonucleoprotein particles are essential ribosome biogenesis factors that function in methylation and processing of ribosomal RNA[1,2,3,4,5,6,7]
We expressed and purified a truncated version of Nop58p, Nop58p_447 from S. cerevisiae fused with the Maltose Binding Protein (MBP) at its N-terminus (MBP-Nop58p_447), for stability reasons
Previous studies showed that this construct did not disrupt the function of box C/D snoRNP34
Summary
Box C/D type of small nucleolar ribonucleoprotein (snoRNPs) particles are essential ribosome biogenesis factors that function in methylation and processing of ribosomal RNA (rRNA)[1,2,3,4,5,6,7]. Tah1p plays a less role in contacting Rvb1/2p but is believed to mediate binding of the molecular chaperone heat shock protein 90 (Hsp90) through its N-terminal TPR (the Tetratricopeptide repeats) motifs[27,30,31]. In these studies, the hexameric Ruv1/2p ring can be resolved at an atomic resolution, structural characterization of the other components has been difficult owing to their flexibility[28,29]. Study of human R2TP complex has made significant progress, it faces the same challenge in characterizing the PIH1D1-RPAP3 (Pih1p-Tah1p homolog) structure[32,33]. In light of the previously known client free R2TP structures both from human and yeast, we are able to place Nop58p at the top of the R2TP complex in a manner consistent with all experimental data
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