Annika Krüger

Sex-chromosome dosage poses a challenge for heterogametic species in maintaining the proper balance of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage asymmetries in avian cells. Through comprehensive allele-resolved multiome analyses, we examine dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Our data reveal that females upregulate their single Z chromosome through increased transcriptional burst frequency, mirroring mammalian X upregulation. Z-protein levels are further balanced in females through enhanced translation efficiency. Additionally, we present a global analysis of promoter elements regulating transcriptional burst kinetics in birds, revealing evolutionary conservation of the genomic encoding of burst kinetics between birds and mammals. Our study provides insights into the regulation of avian dosage compensation, and when considering all regulatory layers collectively, an unexpected similarity between avian and mammalian dosage compensation becomes apparent.

Accurate protein synthesis depends on the proper recognition of stop codons by release factors (RFs). The human mitochondrial genome deviates from the standard genetic code by using not only the canonical stop codons UAG and UAA but also two non-canonical stop codons, AGA and AGG. Consequently, human mitochondria employ two release factors—mtRF1a and mtRF1—that recognize canonical and non-canonical stop codons, respectively. Surprisingly, mtRF1 is conserved across vertebrates, despite the absence of non-canonical stop codons in many species. Here, we investigate the function of mtRF1 in Mus musculus, whose mitochondrial open reading frames (mt-ORFs) lack non-canonical stop codons. Using a combination of biochemical approaches and mitoribosome profiling, we demonstrate that mtRF1 retains functional activity as a non-canonical RF, mediating an alternative release mechanism for COX1 via stop codon readthrough. Similarly, in human mitochondria, AGG stop codon readthrough enables ND6 release by mtRF1a in the absence of mtRF1. Our data reveal that mitoribosome stop codon readthrough is a conserved mechanism that compensates for the loss of RFs. While this mechanism facilitates access to alternative stop codons, it is also associated with RNA degradation. A targeted siRNA screen shows that components of the mitochondrial degradosome—including PNPase and SUV3—as well as the mitochondrial poly(A) polymerase MTPAP, participate in this decay pathway. Together, our study provides new insights into mitochondrial translation termination and RNA quality control, revealing stop codon readthrough as a rescue mechanism that enables the completion of mitochondrial protein synthesis when standard termination fails.

Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency similar to mammalian X-upregulation, and that Z-protein levels are further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species.

Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.

Mitochondrial translation is a complex process responsible for the synthesis of essential proteins involved in oxidative phosphorylation, a fundamental pathway for cellular energy production. Central to this process is the termination phase, where dedicated factors play a pivotal role in ensuring accurate and timely protein production. This review provides a comprehensive overview of the current understanding of translation termination in human mitochondria, emphasizing structural features and molecular functions of two mitochondrial termination factors mtRF1 and mtRF1a.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.

Mammalian mitochondrial ribosomes (mitoribosomes) synthesize a small subset of proteins, which are essential components of the oxidative phosphorylation machinery. Therefore, their function is of fundamental importance to cellular metabolism. The assembly of mitoribosomes is a complex process that progresses through numerous maturation and protein-binding events coordinated by the actions of several assembly factors. Dysregulation of mitoribosome production is increasingly recognized as a contributor to metabolic and neurodegenerative diseases. In recent years, mutations in multiple components of the mitoribosome assembly machinery have been associated with a range of human pathologies, highlighting their importance to cell function and health. Here, we provide a review of our current understanding of mitoribosome biogenesis, highlighting the key factors involved in this process and the growing number of mutations in genes encoding mitoribosomal RNAs, proteins, and assembly factors that lead to human disease.

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a ‘PAR code’.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD⁺ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3’ phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD⁺ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.