Marlene Oeffinger

RNA editing in mitochondria is vital for many eukaryotes. In plants, mitochondrial C-to-U deamination editing is catalyzed by tens to hundreds of PPR-DYW proteins, each typically dedicated to a specific site. Where retained, PPR-DYW family expanded independently in all eukaryotic groups examined here -- except in marine microeukaryotes diplonemids, which encode a single homolog despite deaminating their mitochondrial RNA at more than 100 sites. Here we characterize this unconventional deaminase, PPRD1, from Diplonema papillatum. Native affinity pulldown of the protein identified 20 predominantly sub-stoichiometric partners, including potential RNA-binding helical-repeat proteins. Among them, the divergent PolX-like protein DAPX1 consistently and reciprocally co-purified with PPRD1 in near-equal proportions. Structural modelling suggests that DAPX1 may stabilize the deaminase catalytic domain and expand its interaction interface. Silencing either PPRD1 or DAPX1 inhibited cell growth and reduced in vivo not only C-to-U, but also A-to-I deamination across five mitochondrial RNA-editing clusters encompassing 110 sites. Together, these results support a model of PPRD1 and DAPX1 forming the core of the Diplonema deamination-editing machinery, with sub-stoichiometric partners acting as specificity factors that guide accurate RNA editing with minimal off-target effects.

Senescent cells, which accumulate with age, exhibit a pro-inflammatory senescence-associated secretory phenotype (SASP) that includes the secretion of cytokines, lipids, and extracellular vesicles (EVs). Here, we established an in vitro model of senescence induced by Raf-1 oncogene in RAW 264.7 murine macrophages (MΦ) and compared them to senescent MΦ found in mouse lung tumors or primary macrophages treated with hydrogen peroxide. The transcriptomic analysis of senescent MΦ revealed an important inflammatory signature regulated by NFkB. We observed an increased secretion of EVs in senescent MΦ, and these EVs presented an enrichment for ribosomal proteins, major vault protein, pro-inflammatory miRNAs, including miR-21a, miR-155, and miR-132, and several mRNAs. The secretion of senescent MΦ allowed senescent murine embryonic fibroblasts to restart cell proliferation. This antisenescence function of the macrophage secretome may explain their pro-tumorigenic activity and suggest that senolytic treatment to eliminate senescent MΦ could potentially prevent these deleterious effects.

The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ATM-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA damage response in human cells. We further show the only UFM1-E3-ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage, and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. A first-ever analysis of nuclear and nucleolar UFMylation targets in response to DSBs induction further identified key DNA repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provides the first evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

For over 40 years, ribosomes were considered monolithic machines that translate the genetic code indiscriminately. However, over the past two decades, there have been a growing number of studies that suggest ribosomes to have a degree of compositional and functional adaptability in response to tissue type, cell environment and stimuli, cell cycle or development state. In such form, ribosomes themselves take an active part in translation regulation through an intrinsic adaptability provided by evolution, which furnished ribosomes with a dynamic plasticity that confers another layer of gene expression regulation. Yet despite the identification of various sources that give rise to ribosomal heterogeneity both at the protein and RNA level, its functional relevance is still debated, and many questions remain. Here, we will review aspects, including evolutionary ones, of ribosome heterogeneity emerging at the nucleic acid level, and aim to reframe ribosome ‘heterogeneity’ as an adaptive and dynamic process of plasticity.The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.