hUTP24 is essential for processing of the human rRNA precursor at site A₁, but not at site A₀

Abstract

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domaincontaining endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A 0 , A 1 , and A 2 . We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5 0 -end of the 18S rRNA segment (site A 1 ). Interestingly, and in contrast to yeast, site A 0 located upstream of A 1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A 1 cleavage site.

Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway

“…Results are slightly different when the Utp24-D68N PINc mutant is expressed. According to previous observations (13), Utp24 D68N is enzymatically deficient but is probably still associated with an assembled SSU processome (40). In these conditions, A 0 is accurately cleaved whereas A 1 and A 2 are blocked leading to the accumulation of the 22S pre-rRNA, an intermediate extending from A 0 to A 3 .…”

Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

“…Processing of the precursors to 28S rRNA, detected by the ITS2 probe, was not significantly affected by depletion of any of the studied factors. Intriguingly, the observed defects in pre-rRNA maturation indicate reduced efficiency of endonucleolytic cleavage at the sites A0, 1 and E, an activity attributed to the SSU processome complex (18,53,54), and impaired processing of the ITS1 region (Figure 8D) (38,53,55). Similar to depletion of ANN complex members, downregulation of FBL, which is part of both the SSU processome and other snoRNPs (21,56), induced a strong accumulation of a 30S-sized precursor.…”

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

“…The PIN endonuclease hUTP24 is responsible for cleavages at sites 1 and 2a (6,7), but the identity of the enzyme that cleaves at site A0 remains unknown. To investigate a potential catalytic function of human hUTP23 in site A0 cleavage, and to understand the role of its conserved Zinc finger motif, we established an RNAi-rescue system for hUTP23 in HEK293 cells.…”