Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart

Milenkovic, Ivan; Santos Vieira, Helaine Graziele; Lucas, Morghan C.; Ruiz-Orera, Jorge; Patone, Giannino; Kesteven, Scott; Wu, Jianxin; Feneley, Michael; Espadas, Guadalupe; Sabidó Aguadé, Eduard, 1981-; Hübner, Norbert; van Heesch, Sebastiaan; Völkers, Mirko; Novoa, Eva Maria

doi:http://dx.doi.org/10.1093/nar/gkad121

Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart

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Milenkovic I, Santos Vieira HG, Lucas MC, Ruiz-Orera J, Patone G, Kesteven S, Wu J, Feneley M, Espadas G, Sabidó E, Hübner N, van Heesch S, Völkers M, Novoa EM. Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart. Nucleic Acids Res. 2023;51(11):5301-24. DOI: 10.1093/nar/gkad121

Abstract

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.