Abstract
Purpose: :
Mutations in WDR36 (WD repeat domain 36) have been identified as causative for GLC1G-linked glaucoma (Monemi et al., HMG 2005). WDR36 encodes for a 951 aa protein characterized by G-beta WD-40 repeats and an UTP-21-domain. Yeast Utp21 is essential for nucleolar processing of 18S rRNA and zebrafish that are deficient in Wdr36 show defects in 18S rRNA processing (Skarie and Link, HMG 2008). To identify the function of WDR36 in mammals, we generated and characterized mutant mice deficient in Wdr36. In addition, the function of WDR36 was analyzed in human trabecular meshwork cells (HTM-N).
Methods: :
Mutant mice that are deficient in Wdr36 were generated by homologous recombination in embryonic stem cells using gene targeting technology resulting in deletion of the start ATG and the first 16 exons. The phenotype of heterozygous (+/-) and homozygous (-/-) embryos was analyzed. To investigate early stages of development, zygotes were isolated and cultivated up to the blastocyst stage. A nested PCR protocol was designed to genotype those early embryonic stages. The expression of Wdr36 was knocked down in wild-type zygotes by microinjection of specific siRNA into one of the pronuclei or the cytoplasm/ooplasm. Specific siRNA was also used to knock down the expression of WDR36 in a HTM-N cell line. Effects of siRNA experiments were analyzed by Northern blot hybridization and experiments involving metabolic labelling (pulse-chase).
Results: :
Embryos homozygous for the null mutation (-/-) were not observed at blastocyst stage or later embryonic stages. In culture, Wdr36-/- embryos did not reach blastocyst stage while no differences in early embryonic development were observed between Wdr36+/- and wild-type embryos. Injection of siRNA in wild-type zygotes resulted in a marked retardation of early embryonic development, and only 10% of injected embryos reached blastocyst stage. In contrast, development of zygotes injected with scrambled siRNA was not obviously different from that of non-injected controls. Following knock down of Wdr36 in HTM-N cells, a substantial decrease of 21S rRNA, the direct precursor of 18S rRNA, was observed by Northern Blot analysis. Metabolic labelling experiments indicate a delay in 18S rRNA maturation.
Conclusions: :
WDR36 is involved in nucleolar processing of 18S rRNA in mammalian cells, and is critically required for early mammalian embryonic development.
Keywords: transgenics/knock-outs • ribosomal RNA • RNAi