Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1

Abstract

The function of RNA is subtly modulated by post-transcriptional modifications. Here, we report an important crosstalk in the covalent modification of two classes of RNAs. We demonstrate that yeast Kre33 and human NAT10 are RNA cytosine acetyltransferases with, surprisingly, specificity toward both 18S rRNA and tRNAs. tRNA acetylation requires the intervention of a specific and conserved adaptor: yeast Tan1/human THUMPD1. In budding and fission yeasts, and in human cells, we found two acetylated cytosines on 18S rRNA, one in helix 34 important for translation accuracy and another in helix 45 near the decoding site. Efficient 18S rRNA acetylation in helix 45 involves, in human cells, the vertebrate-specific box C/D snoRNA U13, which, we suggest, exposes the substrate cytosine to modification through Watson–Crick base pairing with 18S rRNA precursors during small subunit biogenesis. Finally, while Kre33 and NAT10 are essential for pre-rRNA processing reactions leading to 18S rRNA synthesis, we demonstrate that rRNA acetylation is dispensable to yeast cells growth. The inactivation of NAT10 was suggested to suppress nuclear morphological defects observed in laminopathic patient cells through loss of microtubules modification and cytoskeleton reorganization. We rather propose the effects of NAT10 on laminopathic cells are due to reduced ribosome biogenesis or function.

Specialized box C/D snoRNPs act as antisense guides to target RNA base acetylation

“…18S rRNA of eukaryotes contains two acetylated cytidine residues, one in helix 34 that is vital for translation fidelity, and another one in helix 45 that constitutes the decoding site of the ribosome (Fig 1A) [18,19]. Acetylation of both cytosines is catalyzed by the highly conserved acetyltransferase Kre33/NAT10 [18–20].…”

“…Recently, we and others identified and characterized two highly conserved acetylated cytosines in helix 34 and helix 45 of yeast, plant and human 18S rRNA along with their corresponding acetyltransferase—Kre33 (yeast) /NAT10 (human) [18–20]. Kre33 assembles with the pre-rRNA when transcription of the 3′ minor domain (encompassing helices 44–45) has been completed and was proposed to bind as a homodimer [21–23].…”

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

“…NGDN has been originally implicated in translational control in neuronal cells (47), but belongs to the ‘U three protein’ (UTP) 3 protein family of SSU processome components by sequence similarity. In addition to NOL10 and NGDN, other nucleolar factors were detected in the MS analysis, including the predicted SSU processome component NAT10 (21), an RNA cytidine acetyltransferase already linked to 40S ribosomal subunit biogenesis (31,48,49). …”

“…NAT10 and eIF4A3 have been previously linked to 40S subunit synthesis and rRNA processing in mammalian cells (20,22,31,48,49,61). While the association of NAT10 with AATF, NGDN and NOL10 was verified by TAP, NAT10 is unlikely to be a core constituent of the ANN complex since neither NAT10 protein levels nor its localization changed upon AATF, NOL10 or NGDN depletion.…”

Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay

“…Nucleosides for RP-HPLC were prepared as described by Gehrke and Kuo [18] and adapted for rRNA as described previously [23]. For the preparation of nucleosides, only purified and intact 18S (100 pmoles) and 25S (60 pmoles) rRNA were used.…”