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, 145 (3), 383-397

Ribosome-mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning

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Ribosome-mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning

Nadya Kondrashov et al. Cell.

Abstract

Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue-specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged; however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development.

Figures

Figure 1
Figure 1. The Rpl38 gene is mutated in Ts, Tss, and Rbt mice
(A) Ts/+ embryos show a midline facial cleft (black dotted line) and eye defects such as a failure in optic fissure closure (black arrow) at E13.5. Ts/+ embryos have an abnormally short and kinky tail (white arrow) by E12.5. (B) An example of the axial skeletal patterning defects in Ts/+ embryos at E18.5. Note the presence of 14 (red circle) instead of 13 ribs in Ts/+ embryos, indicating that the first lumbar vertebra that normally does not possess a rib anlage is now transformed anteriorly into a thoracic vertebra. The rib cage has been removed in these photographs. (C) The exon-intron organization of the Rpl38 gene. The region deleted in the Ts mutation is illustrated on the genome structure. The coding region of the Rpl38 gene is shadowed. The initiation codon ATG for the first methionine is located at the end of exon2. PCR and Southern Blot analysis of the genome structure of the Rpl38 locus in Ts mice with the indicated primers (Sp102 and Sp99) and probes (Probe 1 and Exon 4) is shown in Fig. S1B. (D) Rescue of Ts skeletal phenotypes in Ts/+; pCAGGS-Rpl38 mice. (E) Rpl38 mutations in Rbt and Tss. In exon3 of Tss, a deletion of a base A in exon3 (arrowhead). Rbt has a C to G transversion mutation at intron2 adjacent to the acceptor splice site near exon3 (arrowhead). (F) In Ts/+ Tss/+, Rbt/+ mice the expression of Rpl38 mRNA is reduced to almost half that of wild-type levels. See also Figure S1.
Figure 2
Figure 2. Homeotic transformations and skeletal patterning defects in Ts/+ embryos
(A-H) Alizarin red and Alcian blue staining of WT and Ts/+ skeletons at E18.5. The frequency of patterning defects is presented in the table on the right (note that not all of these phenotypes are shown). Lateral view of the cervical (C) and upper thoracic (T) region. In Ts/+ embryos the anterior tuberculum (AT) normally a feature of C6 is instead present on C5. The star indicates an ectopic, partially formed rib on C6 in Ts/+ embryos. The black arrow indicates the presence of ribs (R) on the first thoracic vertebrae in Wt embryos that is a feature of C7 in Ts/+ embryos. Fusions between cervical vertebrae in Ts/+ embryos (white arrow). (C-D) Ventral view of the thorax reveals that in Ts/+ embryos there are eight pairs of vertebrosternal ribs instead of seven attached to the sternum (yellow arrow heads). The attachment site of ribs to the sternum is asymmetric in Ts/+ embryos resulting in a crankshaft sternum. (E-F) In the lumbar region, an extra pair of ribs (black asterisks) is present on the first lumbar vertebrae in Ts/+ embryos. A normal number of lumbar segments in Ts/+ embryos suggest that a partial transformation of S1 into a lumbar identity (S1*) has occurred. (G-H) Sacral vertebrae in Wt (G) and Ts/+ (H). In Wt embryos, the first three sacral vertebrae are fused to make the sacral bone (SB), while in Ts/+ embryos, the fourth and sometimes fifth sacral vertebrae are fused (red arrow heads). See also Figure S2.
Figure 3
Figure 3. Analysis of global protein synthesis rates and polysome profiling within the neural tube and somites of Wt and Ts/+ embryos
(A) Transgenic mice expressing a stable, genetically encoded translational reporter for cap- and IRES-dependent translation (CMV-HCV-IREST) were intercrossed with Ts/+ mice. No changes in CAP or IRES-dependent translation are evident within the neural tube and somites of Ts/+ embryos compared to WT. (B) Overlay of polysome profiles from Wt (red solid line) and Ts/+ (blue dotted line) somite stage 40 embryos (~E11.0). Schematic of micro-dissection strategy is shown (insert). Neural tube (grey) and somites (orange) were simultaneously microdissected as illustrated (red dashed line). (C) qPCR analysis of 8/39 Hox mRNAs that show differential association in polysome fractions (fraction numbers are depicted on the bottom of the graph) in Ts/+ tissue fragments, (n=5) for each genotype. The bottom two graphs are representative examples of 2/31 Hox mRNAs that do not show changes in polysome association in Ts/+ tissue fragments. P values (Student's t-test) for each polysome fraction are shown. Data are presented as the average ± SEM. See also Figure S3.
Figure 4
Figure 4. Analysis of Hox gene mRNA expression levels, boundaries, and protein levels in Wt and Ts/+ embryos compared to additional RP mouse deficiencies
(A-O) Somite stage 40 (~E11.0) Wt and Ts/+ embryos were analyzed. Unless specified, all experiments have been performed utilizing the microdissected neural tube & somite tissue fragment, Western Blot (WB), whole mount in situ hybridization (WISH). (A) Hoxa5 qPCR. (B) Hoxa5 WISH showing the anterior boundary of expression. (C) HOXA5 WB. (D) HOXA5 WB in microdissected neural tube (NT) or Somites (S). (E) HOXA5 immunofluoresence (grey scale) of Wt and Ts/+ transverse tissue sections at the forelimb level. Neural tube (top panel) and somite (bottom panel) expression is shown from serial sections of the same embryo (10X). Dorsal root ganglion (drg). Quantification of mean fluorescence intensity is shown in the graphs to the right. (F) Quantification of florescent intensities of HOXA5 within individual cells (ticks on the X-axis) of the NT. (G) Hoxa11 qPCR. (H) Hoxa11 WISH showing the anterior boundary of expression. (I) HOXA11 WB. (J) Hoxb13 qPCR. (K) Hoxb13 WISH. (L) HOXB13 WB. (M) Hoxc4 qPCR. (N) Hoxc4 WISH showing the anterior boundary of expression. (O) HOXC4 WB. (P) Relative [S35] methionine incorporation to monitor the rates of de-novo protein synthesis in the neural tube and somites of stage 40 embryos with distinct RP deficiencies (see also Figure S4D). The graph shows quantification in n=3 embryos, *P=0.04, **P=0.007, ***P=0.03 (Student's t-test), not significant (n.s.). (Q) HOXA5 WB, a target mRNA translationally deregulated in Ts/+ embryos (see C-D), reveals no change in expression levels in other RP deficiencies. Data are presented as the average ± SEM. See also Figure S4.
Figure 5
Figure 5. Comparison of the Ts/+ axial skeletal and neural tube patterning defects with Hox loss of function mutants
(A) Schematic representation of the axial skeleton of WT and Ts/+ mice. Illustrated in red are transformations or patterning defects frequently observed in Ts/+ mice. The red shadow outlining the sternum indicates abnormal rib to sternum attachment in Ts/+ mice. Corresponding Hox mutants that show similar phenotypes are listed in boxes to the right of the Ts/+ axial skeleton schematic representation: Hoxc8 enhancer element knockout (Hoxc8EE)(Juan and Ruddle, 2003), Hoxa5 knockout (Hoxa5)(Jeannotte et al., 1993), Hoxa4 knockout (Hoxa4)(Horan et al., 1994), Hoxc8 knockout (Hoxc8)(Le Mouellic et al., 1992), Hoxa9 knockout (Hoxa9)(Fromental-Ramain et al., 1996), Hoxd10 knockout (Hoxd10)(Carpenter et al., 1997), Hoxa11 knockout (Hoxa11)(Small and Potter, 1993). See also Figure 2. (B) Analysis of Pea3 expression at the Branchial Level (BL) of the neural tube in serial transverse sections (BL1-BL4; caudal to rostral) at somite stage 40. The bottom sections show the ventral left quadrant of the neural tube that is outlined with a dashed white line. The graph shows quantification of Pea3+ LMC motor neurons (MNs) from n=7 WT and Ts/+ embryos and P values (Student's t-test) are indicated. (C) Islet 1 expression at the Branchial Level (BL). The bottom sections show the ventral left quadrant of the neural tube that is outlined with a dashed white line. No differences in Islet 1 expression are observed in Ts/+ embryos. See also Figure S5.
Figure 6
Figure 6. RPL38 controls 80S complex formation on selective Hox mRNAs from the ribosome
(A-B) 80S monosome–mRNA complex formation assessed in-vivo in microdissected neural tube & somites from somite stage 40 WT and Ts/+ embryos, n=5. qPCR analysis of Hox mRNAs from fractions 7-8 of sucrose gradients, corresponding to the 80S monosome, shows diminished complex formation on specific Hox mRNAs found to be decreased at the translation level in Ts/+ embryos (A) but not others (B). P values (Student's t-test) are shown. (C) Two representative models for control of 80S complex formation by RPL38, on or off the ribosome. (D-E) Neural stem cells (NSC), the murine embryonic mesenchymal cell line C3H/10T1/2 (10T1/2), microdissected neural tube & somites from E11.5 embryos, and embryonic stem cell lines treated with Retinoic Acid for the indicated amount of time (hours) were fractioned on a sucrose cushion to separate the ribosomal fraction from ribosome- free cytosol and both fractions were subject to immunoblot analysis. (F-G) Sucrose gradient fractionation of neural tube & somites from E11.0 embryos and subsequent Western Blot analysis with representative small and large RPs. Note that Rpl38 is only present in fractions containing the 60S, 80S, and polysomes. (G) Upon puromycin (Puro) treatment that specifically releases nascent polypeptides from ribosomes and dissociates ribosomal subunits, RPL38 is only found associated with the 60S and is localized to the same fractionations as another RP belonging to the large subunit. Data are presented as the average ± SEM. See also Figure S6.
Figure 7
Figure 7. Ribosomal protein expression during organogenesis
(A-C) RPL38 in situ hybridization on tissue sections at E11.5. (A) Rpl38 expression in the developing mouse face. Enriched RPL38 expression is observed in the medial nasal process (mns), mandibular component of the first branchial arch (mc), cerebellar plate (cp), diencephalon (di), midbrain (mb). Insert is from a more lateral serial section of the eye from the head region showing that RPL38 expression is enriched in the neural retina (nr). Third ventricle (ven), Lens vesicle (lv). (B) Sagittal section showing markedly enriched RPL38 expression in all A-P somite derivates (black arrowhead). The upper and lower panels are representative serial sections of the same embryo at more anterior (a) and posterior (p) positions. (C) Transverse tissue section of the neural tube (outlined in a dotted line) at the brachial level. To the right is a serial section stained with a HOXA5 antibody. The overlay of RPL38 and HOXA5 expression is shown in the bottom panel, where strong overlap is observed in motor neurons within the lateral motor column (LMC). (D) Heatmap diagram displaying the hierarchical clustering of the expression of 72 ribosomal proteins. The columns in the diagram are separated by tissue and primary cell type; each row is a ribosomal protein, large (l) or small (s) subunit. The color bar at the top indicates the color-coding of gene expression from +4 to -4 in log2 space. (E) Proposed model of RP specificity in control of gene expression during embryogenesis. The enriched expression of specific ribosomal proteins (RP) in different tissues may confer translational specificity to distinct classes of mRNAs (a,b,c). Brain (green), Limbs (red), Somites (blue). (See also Table S3)

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