Highlights d The PIDDosome controls hepatocyte ploidy during postnatal development & regeneration d The PIDDosome defines the speed of liver regeneration posthepatectomy d Aneuploidy in the liver correlates with basal ploidy state but is not limited by CASP2 d E2F family members regulate expression of CASP2 and PIDD1 for liver ploidy control

the primary miR-15a stem-loop structure per se is a poor Microprocessor substrate• cleavage of pri-miR-15a requires the processing of an additional miRNA stem-loop on the same RNA• sequential pri-miRNA processing or "cluster assistance" is mediated by SAFB proteins • SAFB2 associates with the Microprocessor .

Highlights• the primary miR-15a stem-loop structure per se is a poor Microprocessor substrate• cleavage of pri-miR-15a requires the processing of an additional miRNA stem-loop on the same RNA • sequential pri-miRNA processing or "cluster assistance" is mediated by SAFB proteins • SAFB2 associates with the Microprocessor Summary MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally silence most protein-coding genes in mammals. They are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent units. Contrasting this view, we here report an unexpected mode of processing of a bicistronic cluster of the miR-15 family, miR-15a-16-1. We find that the primary miR-15a stem-loop is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage, and describe SAFB2 as a novel accessory protein of DROSHA. Notably, SAFB2-mediated cluster assistance expands to other clustered pri-miRNAs including miR-15b, miR-92a and miR-181b, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal DGCR8/DROSHA substrates in clustered primary miRNA transcripts.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.Electronic supplementary materialThe online version of this article (10.1007/s00018-017-2700-0) contains supplementary material, which is available to authorized users.