The ribosome biogenesis factor yUtp23/hUTP23 coordinates key interactions in the yeast and human pre-40S particle and hUTP23 contains an essential PIN domain.

Graeme R Wells,Claudia Schneider,Franziska Weichmann,Katherine E Sloan,Nicholas J Watkins,David Colvin

doi:10.1093/nar/gkw1344

Abstract

Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using in vivo RNA–protein crosslinking and gel shift experiments, we reveal that yUtp23/hUTP23 makes direct contacts with expansion sequence 6 (ES6) in the 18S rRNA sequence and that yUtp23 interacts with the 3΄ half of the snR30 snoRNA. Protein–protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation.

Highlights

Eukaryotic ribosomal (r)RNAs are processed from an initial 35S (Saccharomyces cerevisiae) or 47S (Homo sapiens) precursor by a series of endonucleolytic cleavages followed by exonucleolytic trimming, which results in the concomitant removal of external (5 ETS, 3 ETS) and internal (ITS1, ITS2) transcribed spacer sequences (Figure 1 and Supplementary Figure S1) [1]
We present data suggesting that the yeast yUtp23snR30 and human hUTP23-U17 complexes may act as hubs to coordinate the binding and release of factors at the 18S expansion sequence 6 (ES6) region
We show that the PIN domain active site amino acid, D31, is needed for pre-rRNA processing suggesting that hUTP23 may be an endonuclease

Summary

Introduction

Eukaryotic ribosomal (r)RNAs are processed from an initial 35S (Saccharomyces cerevisiae) or 47S (Homo sapiens) precursor (pre-rRNA) by a series of endonucleolytic cleavages followed by exonucleolytic trimming, which results in the concomitant removal of external (5 ETS, 3 ETS) and internal (ITS1, ITS2) transcribed spacer sequences (Figure 1 and Supplementary Figure S1) [1]. The early pre-rRNA cleavages, at sites A0, A1 and A2 in yeast and A’, A0, A1/1 and 2a/E in humans, are critical for 18S rRNA maturation and require the small subunit (SSU) processome, a large ribonucleoprotein complex [2]. The U3 small nucleolar (sno)RNA, a key component of the SSU processome, base-pairs with the 5 ETS and 18S rRNA sequences to guide the formation of the conserved central pseudoknot, which is an essential feature of the 40S ribosomal subunit [2,3]. The PIN (PilT N-terminus) endonucleases yUtp24/Fcf (hUTP24 in humans) and yNob (hNOB1 in humans) are critical for 18S rRNA processing. Pre-rRNA cleavages at three sites, A0, A1/1 and A2/2a, require the presence of yUtp24/hUTP24 and the PIN endonuclease domain of the protein is essential for cleavages at A1/1 and A2/2a [4,5,6,7].

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Results

Conclusion