WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N⁷-methylation of G1639 in human 18S rRNA

Abstract

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2 ′ -O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in WilliamsBeuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3 ′ -extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3 ′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3 ′ -RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N 7 -methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.

The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

“…In agreement with this hypothesis, investigators have observed retention of fluorescently labeled pre-40S in the nucleus upon WBSCR22 depletion (Wild et al. , 2010), and the 3′-extended forms of 18S-E that accumulates under these conditions are reported to be nuclear (Haag et al. , 2015).…”

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…For generation of tetracycline‐inducible stable cell lines the NSUN3 or ABH1 CDS were cloned into the pcDNA5 vector with C‐terminal GFP or His‐PreScission protease cleavage site‐2×FLAG (HisPrcFlag) tag. The catalytically inactive NSUN3 C265A mutant was generated by site‐directed mutagenesis (Haag et al , 2015b). The constructs were transfected into HEK293 Flp‐In T‐Rex cells according to the manufacturer's instructions and as described (Sloan et al , 2015).…”

“…The ABH1 D233A and R233A, and NSUN3 C265A mutants were generated by site‐directed mutagenesis (Haag et al , 2015b). Recombinant proteins were expressed in Escherichia coli (DE3) Rosetta pLysS (NSUN3, ABH1) or (BL21) Codon Plus (MTIF2, TUFM) cells and details of protein purification are given in the Appendix Supplementary Methods.…”

“…The coding sequences of human NSUN3, ABH1 or FTO were cloned into a pQE80 derivative encoding an N-terminal His 14 -MBP-tag (Weis et al, 2014) and the CDS of MTIF2 or TUFM into a pQE80 derivative encoding a C-terminal His 10 -tag (Mingot et al, 2004). The ABH1 D233A and R233A, and NSUN3 C265A mutants were generated by site-directed mutagenesis (Haag et al, 2015b). Recombinant proteins were expressed in Escherichia coli (DE3) Rosetta pLysS (NSUN3, ABH1) or (BL21) Codon Plus (MTIF2, TUFM) cells and details of protein purification are given in the Appendix Supplementary Methods.…”

NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner

“…A very recent report has shown that yeast and worm homologs of human NSUN5 methylate C2278 in 25S rRNA of Saccharomyces cerevisiae and C2381 in 26S rRNA of Caenorhabditis elegans; in addition, the reduction in the levels of NSUN5 homologs decreases translational fidelity in yeast and increases the lifespan of S. cerevisiae, C. elegans and Drosophila melanogaster (Schosserer et al, 2015). The human ortholog of yeast Bud23, WBSCR22 (Merm1), has also been shown to mediate N 7 -methylation of G1639 and 18S rRNA maturation in cells (Haag et al, 2015); however, little is known about the base methyltransferase of rRNA in mammals.…”