Abstract
Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.
Highlights
Ribosomes are essential nanomachines responsible for protein production in every cell
We have investigated systematically by RiboMethSeq the complete 2’-O methylation repertoire of rRNAs in developing frog embryos, confirming previously mapped positions, identifying new ones, and revealing that specific sites are strongly hypomodified at late stages of embryogenesis
We analyzed the spatial expression of the selected genes by whole-mount in situ hybridization (WISH)
Summary
Ribosomes are essential nanomachines responsible for protein production in every cell. They are sophisticated ribonucleoprotein particles consisting of two subunits of unequal size with specialized functions in translation. Ribosome biogenesis entails numerous reactions, including synthesis of the components (four ribosomal RNAs (rRNAs) and eighty ribosomal proteins in human ribosomes) and their modification, transport, and assembly into functional subunits [2,3]. These reactions are aided by numerous trans-acting factors that interact transiently with the maturing ribosomal subunits [4,5]. Surveillance mechanisms monitor the faithful production of ribosomal subunits, cells can produce ribosomes that differ in composition and possibly in function [6,7]
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