Griffin JN et al. (2018), RPSA, a candidate gene for isolated congenital ...

XB-ART-55377

Development 2018 Oct 18;14520:. doi: 10.1242/dev.166181.

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RPSA, a candidate gene for isolated congenital asplenia, is required for pre-rRNA processing and spleen formation in Xenopus.

Griffin JN , Sondalle SB , Robson A , Mis EK , Griffin G , Kulkarni SS , Deniz E , Baserga SJ , Khokha MK .

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A growing number of tissue-specific inherited disorders are associated with impaired ribosome production, despite the universal requirement for ribosome function. Recently, mutations in RPSA, a protein component of the small ribosomal subunit, were discovered to underlie approximately half of all isolated congenital asplenia cases. However, the mechanisms by which mutations in this ribosome biogenesis factor lead specifically to spleen agenesis remain unknown, in part due to the lack of a suitable animal model for study. Here we reveal that RPSA is required for normal spleen development in the frog, Xenopus tropicalis Depletion of Rpsa in early embryonic development disrupts pre-rRNA processing and ribosome biogenesis, and impairs expression of the key spleen patterning genes nkx2-5, bapx1 and pod1 in the spleen anlage. Importantly, we also show that whereas injection of human RPSA mRNA can rescue both pre-rRNA processing and spleen patterning, injection of human mRNA bearing a common disease-associated mutation cannot. Together, we present the first animal model of RPSA-mediated asplenia and reveal a crucial requirement for RPSA in pre-rRNA processing and molecular patterning during early Xenopus development.

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Species referenced: Xenopus tropicalis
Genes referenced: dtl hhex nkx2-1 nkx2-5 nkx3-2 pax2 pitx2 rpsa tcf21
GO keywords: rRNA processing [+]
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???displayArticle.omims??? ASPLENIA, ISOLATED CONGENITAL; ICAS
Phenotypes: Xtr Wt + Rpsa MO (Fig. 2 A) [+]

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	Fig. 1. rpsa is dynamically expressed in development. (A,B) Both in situ hybridization (A) and ultra-high temporal resolution RNA-seq analysis (B) reveal low levels of rpsa transcripts prior to stage 12 in X. tropicalis. Expression increases throughout subsequent stages and is primarily associated with the developing neural folds (nf), brain (b), cranial neural crest (cnc) and ventral mesoderm/blood islands (vm/bl). Expression is also diffusely detected in the lateral mesoderm (lm) where the spleen will form. pa, pharyngeal arches.
	Fig. 2. Depletion of Rpsa using either a translation (RpsaATG) or splice (RpsaSplice) blocking morpholino impairs expression of nkx2-5 and bapx1 in the spleen of stage 38 rpsa morphants. (A) Representative images of the expression phenotype. Middle and right panels are magnifications of the spleen region marked by a box in the left panel. Black arrowheads indicate normal expression. Blue arrowheads indicate reduced expression. Red arrowheads indicate lack of expression. (B) The percentage of embryos with reduced or absent nkx2-5 or bapx1 expression in the developing spleen following rpsa depletion. ***Pâ‰¤0.0001 (two-tailed Fisher's exact test).
	Fig. 3. The human R180G mutation is detrimental to function. (A) Representative images of nkx2-5 expression in the spleen anlage of stage 38 embryos. Depletion of Rpsa causes a severe reduction of nkx2-5 expression, which can be ameliorated by injection of human WT RPSA mRNA but not by injection of R180G mutated mRNA. Green arrowheads highlight robust expression; red arrowheads indicate reduced expression. (B,C) The percentage of embryos with reduced or absent nkx2-5 (B) or bapx1 (C) expression. P<0.001, *P<0.0001 (two-tailed Fisher's exact test). CMO, control morpholino; rpsaATG, translation-blocking MO; NS, not significant; UC, uninjected control.
	Fig. 4. Rpsa is required for pre-rRNA processing. (A) Diagram of pre-rRNA processing pathways in X. tropicalis. The primary transcript pre-rRNA undergoes several nucleolytic cleavages to produce the mature 18S, 5.8S and 28S rRNAs. c (red), position of the northern blot probe; ETS, external transcribed spacers; vertical lines indicate cleavage sites. (B-D) Depletion of Rpsa causes pre-rRNA processing defects that are rescued by WT human RPSA (hRPSA) but not by ICA-mutated RPSA mRNA (R180G). Stage 38 X. tropicalis pre-rRNAs isolated from pools of 10-15 whole embryos were detected by northern blotting (B). The 7SL RNA was used as a loading control. +, injection; âˆ’, no injection. (C) RAMP quantitation of the ratios of pre-rRNAs relative to each other. (D) RAMP quantitation of the pre-rRNAs relative to the 7SL loading control. Data are mean+s.d. for four independent biological replicates. Pâ‰¤0.01, Pâ‰¤0.001, ***Pâ‰¤0.0001 (two-way ANOVA with Tukey's multiple comparisons test). CMO, control morpholino; rpsaATG, translation-blocking MO; UC, uninjected control.
	Fig. S1 Rpsa depletion does not affect development of other early developmental processes examined. A) Gross morphology of stage 45 control and rpsa depleted embryos. B) Optical coherence tomography of the craniofacial cartilages of stage 45 tadpoles injected with rpsa MO in one cell at the two cell stage, allowing comparison with the uninjected control side of the embryo. No significant differences were detected. C) Expression of nkx2.1, pax2 and hex is unaffected by depletion of rpsa. D) The laterality gene pitx2c is normally expressed in the left lateral mesoderm of rpsa morphants at stage 28 (green arrowheads).
	Fig. S2 Rpsa depletion impairs spleen development. A) Representative images of reduced bapx1 expression observed in stage 38 rpsa morphants. Panels on right are magnifications of the spleen anlage in the corresponding embryo. B) Timeline of nkx2.5 expression. The percentage of embryos with reduced expression is significantly increased in rpsa morphants at both stage 37 and stage 40. C) Expression of pod1 (capsulin) is disrupted in stage 38 morphants relative to controls (compare red and green arrowheads). Single and triple asterisks indicate P<0.01 and P<0.0001 respectively. UC, uninjected control; CMO, control morpholino; RpsaATG, targeting translation blocking morpholino oligonucleotide.
	Fig. S3 The R180G disease variant. A) Alignment of RPSA amino acid sequence across selected metazoan species. Note that R180 (red box) is conserved. B) Structural model of the R180 residue in WT human RPSA using PyMol. R180 in wild-type human RPSA is shown with its predicted polar contacts with D14 and E181 in RPSA. Orange dotted lines indicate polar contacts. C) Substitution of R180 with a glycine (R180G) is predicted to abolish these interactions, likely destabilizing the proteinâs tertiary structure.
	rpsa (ribosomal protein SA) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior left.
	rpsa (ribosomal protein SA) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, laterall view, anterior left.

References [+] :

Amsterdam, Identification of 315 genes essential for early zebrafish development. 2004, Pubmed