The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Abstract

Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A 1 and͞or A2.ribosome biogenesis ͉ small subunit processome ͉ endonuclease ͉ yeast ͉ RNA processing R ibosomes translate mRNA into proteins and, thereby, govern cell growth and survival. In Saccharomyces cerevisiae, ribosome biogenesis starts with the transcription of the rDNA into a polycistronic rRNA precursor by RNA polymerase I in the nucleolus. The primary transcript, the 35S pre-rRNA, encodes the small subunit (SSU) rRNA, 18S, the large subunit rRNAs, 25S and 5.8S, and also external and internal transcribed spacer regions (5ЈETS, ITS1, ITS2, and 3ЈETS; Fig. 1). This precursor is extensively endoand exonucleolytically cleaved to mature the rRNAs. Maturation begins with endonucleolytic cleavages at sites A 0 , A 1 , and A 2 , the latter separating the 20S pre-rRNA, the direct precursor to the mature 18S rRNA, and the 27SA 2 pre-rRNA, the precursor to the large subunit rRNAs. The 20S pre-rRNA is exported into the cytoplasm as part of the 43S preribosome, where the 18S rRNA is matured by cleavage at site D.The A 0 , A 1 , and A 2 pre-rRNA cleavages require a large ribonucleoprotein complex, the SSU processome͞90S preribosome, which contains at least 40 different proteins and numerous small nucleolar RNAs (snoRNAs) (1-4). This complex likely corresponds to the terminal knobs seen in Miller chromatin spreads (1, 5). Depletion of essential SSU processome components prevents cleavages at sites A 0 -A 2 but not A 3 . Although this results in accumulation of the 23S pre-rRNA and inhibition of 18S synthesis, maturation of the 25S and 5.8S rRNAs can occur normally (Fig. 1).Despite the identification of numerous proteins involved in early pre-18S rRNA processing by large-scale purifications, the endonuclease(s) and possible candidates that carry out these cleavages have remained elusive. Recently, an interesting group of proteins possessing a PIN domain (PilT N terminus, PINc domain in SMART database) has been impl...

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

“…S3A for location of the probes). In line with previous reports, 39,40 we noted that yUtp24 dysfunction (irrespective of whether yUtp24 levels were low or the putative enzymatic activity of the protein was abolished) resulted in a significant decrease of mature 18S rRNA. This was accompanied by a marked reduction of 20S prerRNA (Fig.…”

“…S3B, C), a stable 18S rRNA precursor extending from site A 1 to A 2 . Interestingly, unlike in one of the previous studies, 39 both yUtp24 depletion and exogenous expression of the mutant protein variant led to the accumulation of 23S pre-rRNA (Fig. S3B, C), indicating that the yUtp24 activity may be important for U3 snoRNAdependent processing at sites A 0 , A 1 , and A 2 .…”

“…39,40 Northern blot analyses and pulse-chase RNA labeling experiments revealed that its presence in the cell seemed to be indispensable for cleavage at site A 0 in the 5 0 -ETS, while its putative ribonucleolytic activity appeared to be necessary for processing at sites A 1 and A 2 (see Fig. 3A for positioning of the processing sites in yeast pre-rRNA).…”

“…It was previously suggested that a protein with a PIN nuclease domain, namely yUtp24 (also called Fcf1), might be involved in the endoribonucleolytic cleavage of yeast pre-rRNA at sites A 0 in the 5 0 -ETS and A 1 , thus forming a mature 18S 5 0 -end, and at site A 2 (which was later shown to be processed by a controversial endoribonucleolytic activity of Rcl1 protein, similar to RNA 3 0 cyclases). [39][40][41][42] Nevertheless, yUtp24 has not been shown to have enzymatic activity in vitro, and in vivo analyses, such as primer extensionbased mapping of the processing sites affected by yUtp24 mutation, have not been performed to date. In this study, we analyzed the role of yUtp24 orthologhUTP24 -in pre-rRNA processing in human cells.…”

Rcl1 Protein, a Novel Nuclease for 18 S Ribosomal RNA Production

“…This observation, as well as the sequence homology of Rcl1 to RNA 3Ј-cyclases, and the fact that 2Ј,3Ј-cyclic phosphates are common endonuclease products, led to the proposal that Rcl1 could be the nuclease for A 2 cleavage (7,11), although that proposal has been recently challenged (12), and alternative proposals have been made (13).…”