Fig 2: Phenotypic and molecular characterization of Rpl3l-/- knockout mice. (A) Strategy for generation of Rpl3-/- and Rpl3l-/- mice using the CRISPR/Cas9 system. Rpl3l-/- mice were successfully generated by introducing a 13 bp deletion in exon 5. Rpl3-/- mice have an embryonic-lethal phenotype. See also Supplementary Figure S1. (B) Relative expression levels of Rpl3l (left) and Rpl3 (right) measured using RT–qPCR and normalized to Gapdh. Rpl3 is ubiquitously expressed, while Rpl3l is heart and muscle specific. Rpl3l-/- mice do not express Rpl3l in any of the tissues (n = 3). Statistical significance was assessed using the unpaired t-test (*P <0.05, **P <0.01, ***P <0.001). (C) Immunofluorescence staining of RPL3 and RPL3L in both WT (left) and Rpl3l-/- mice heart tissues (right). Nuclei have been stained with DAPI and are shown in blue; actin is depicted in green and RPL3L (uL3L) (top) and RPL3 (uL3) (bottom) in red. (D) Western blot analysis of RPL3L (uL3L) (left) and RPL3 (uL3) (right) in cardiomyocytes isolated from WT and Rpl3l-/- hearts (n = 3 and n = 6, respectively). At the bottom, barplots depicting the fold change of RPL3 (uL3) and RPL3L (uL3L) expression in cardiomyocytes is shown. RPL3L (uL3) and RPL3 (uL3) levels were normalized to GAPDH. See also Supplementary Figure S4 for full blot images, and Supplementary Figure S5 for western blot results using total heart samples from WT and Rpl3l-/- mice. (E) Representative histological sections of WT and Rpl3l-/- heart tissues stained with H&E. A total of 10 mice were included in the histological analyses. See also Supplementary Figure S9. (F) EchoMRI analyses of aged (55-week-old) WT and Rpl3l-/- mice, in which weight, fat and lean mass were measured for each animal (n = 5). Statistical significance was assessed using unpaired t-test (*P <0.05).

Fig 3: RPL3L (uL3L) is a vertebrate RP paralogue with restricted tissue and developmental expression patterns, such as post-natal expression in mouse cardiomyocytes. (A) Heatmap of mRNA expression levels (log RPKM) of ribosomal proteins and their respective paralogues across embryonic (green: E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5 and E18.5) and post-natal mice tissues (pink: P0, P3, P14, P28 and P63). Processed data (RPKM) were obtained from Cardoso-Moreira et al. (40). See also Supplementary Figure S7 for a heatmap containing all RPs. (B) mRNA expression levels of ribosomal paralogue pairs (RPKM) in three different tissues (brain, liver and heart) for Rpl3/Rpl3l (upper panel), Rpl22/Rpl22l1 (middle panel) and Rpl7/Rpl7l1 (bottom panel). The developmental stages, shown on the x-axis, have been coloured depending on whether they correspond to embryonic (green) or post-natal (pink) stages. (C) Structural alignment of human RPL3 (red) and RPL3L (uL3L) (cyan); the C-terminus is highlighted (left) as is the location of RPL3 (uL3)/RPL3L (uL3L) within the ribosome (right). The C-terminus of RPL3 (uL3) and RPL3L (uL3L) is located at the surface of the ribosome, whereas the N-terminus of both proteins lies closer to the peptidyltransferase centre (PTC). The ribosome structure has been obtained from the cryo-electron microscopy structure of the human 80S ribosome, corresponding to PDB code 6IP5 (101), which includes RPL3. The Homo sapiens RPL3L (uL3L) structure was obtained from the ModBase (102) database and structurally superimposed to the RPL3 structure in the 80S ribosome. (D) T-distributed stochastic neighbour embedding (T-SNE) plot depicting Rpl3l expression across mouse heart cell types. Expression data have been extracted from publicly available single-cell RNA-seq data from Ren et al. (60). Each dot represents a cell. Expression levels are shown as umi (unique molecular identifiers). Abbreviations: CM, cardiomyocytes; EC, endothelial cells; FB, fibroblasts; MP, macrophage; T, T cells.

Fig 4: Pressure overload leads to an increase in Rpl3 expression and a decrease in Rpl3l expression in the heart. (A) The expression of Rpl3 and Rpl3l across cell types in mouse hearts before (week 0) and after hypertrophy (week 8) induced by transverse aortic constriction (TAC). Cardiomyocytes (CM) are circled. Expression levels are shown as umi (unique molecular identifiers). (B) TAC leads to heart hypertrophy that correlates with increased Rpl3 expression and decreased Rpl3l expression. The Rpl3/Rpl3l ratio is significantly increased at all time points (2, 4, 7 and 21 days after surgery) when compared with control hearts. See also Supplementary Figures S19 and S20 and Supplementary Table S15. (C) Effects of TAC-induced hypertrophy on Rpl3–Rpl3l interplay are impaired in Lin28a-/- mice. See also Supplementary Figure S22. (D) Rpl3 (left) and Rpl3l (right) nuclear mRNA levels in cardiomyocytes from the left ventricle (LV), remote myocardium (RM) and border zone (BZ) after myocardial infarction. Processed data (RPKM) were obtained from Günthel et al. (81). (E) Rpl3 and Rpl3l mRNA expression levels at different time points (0 min, 10 min, 1 h, 6 h, 24 h and 72 h) after myocardial infarction. Processed data (RPKM) were obtained from Liu et al. (82). (F) Model showing the Rpl3–Rpl3l interplay in resting (top) and hypertrophic (bottom) conditions. In the resting heart, Rpl3l is predominantly expressed in cardiomyocytes, and the RPL3L protein negatively regulates Rpl3 expression, while RPL3L (uL3L)-containing ribosomes do not establish close contact with mitochondria. Upon hypertrophic stimuli, Rpl3l expression is impaired and Rpl3l mRNA is degraded in the cytoplasm, leading to an increased expression of Rpl3. RPL3 (uL3)-containing ribosomes establish close contact with mitochondria.

Fig 5: RPL3L and RPL3 are incorporated into translating ribosomes in WT and Rpl3l-/- hearts, respectively. (A) Polysome profiles of liver (top), WT heart (middle) and Rpl3l-/- heart (bottom) were performed in 10–50% sucrose gradients with corresponding western blot analyses, in two biological replicates. Two hearts were pooled together per replicate. Membranes were probed with anti-RPL3L (uL3L), anti-RPL3 (uL3) and anti-RPS6 (eS6) antibodies to show the incorporation of both paralogues in translating ribosomes. (B) Polysome profiles fractions of a WT heart (top) and an Rpl3l-/- heart (bottom), analysed with western blot and probed with anti-RPL3L (uL3L), anti-RPL3 (uL3) and anti-RPS6 (eS6). Fractions 8 and 9, corresponding to the monosome peak of the polysome profile, of a Rpl3l-/- heart and WT heart, respectively, were used as positive controls. (C) Puromycin incorporation assay performed on WT and Rpl3l-/- hearts in biological triplicates. GAPDH was used as loading control and for normalization in the densitometric analysis. Statistical significance was assessed using unpaired t-test. Puro stands for puromycin.