Abstract
During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.
Highlights
Human ribosomes are comprised of two ribonucleoprotein subunits: the small subunit (40S) containing one ribosomalRNA and 33 ribosomal proteins, and the large subunit (60S) containing three rRNAs (5S, 5.8S, 28S) and ribosomal proteins [1]
We further investigated how human disease might alter the landscape of rRNA posttranscriptional modifications (PTMs) by assessing the PTMs of rRNAs of the 80S ribosome from patients with familial dyskeratosis congenita (DC) caused by mutations in the gene DKC1, the product of which converts uridine to
To determine the PTMs of human rRNAs in TK6 cells, we applied SILNAS [22] to 5S, 5.8S, 18S, and 28S rRNAs that had been purified by reversed-phase liquid chromatography (LC) [28]
Summary
Human ribosomes are comprised of two ribonucleoprotein subunits: the small subunit (40S) containing one ribosomalRNA (rRNA; 18S) and 33 ribosomal proteins, and the large subunit (60S) containing three rRNAs (5S, 5.8S, 28S) and ribosomal proteins [1]. Each purified rRNA was mixed with an in vitro transcribed G-13 C10 -labeled (guanosine labeled with 10 13 Catoms) reference RNA having the same sequence as each sample RNA; the mixture was digested with RNase T1 and analyzed with a nanoflow LC-coupled MS system equipped with Ariadne software to identify each RNA fragment and to detect modified oligonucleotides (for details, see Supplementary Figures S2 and S3).
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