Mitochondria fulfill central functions in metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, central mechanisms underlying mitochondrial gene expression remain enigmatic. A lack of suitable technologies to target mitochondrial protein synthesis in cells has limited experimental access. Here, we silenced the translation of specific mitochondrial mRNAs in living human cells by delivering synthetic peptide-morpholino chimeras. This approach allowed us to perform a comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and provides a strategy to address mitochondrial gene expression in living cells. The approach can potentially be used to analyze mechanisms and pathophysiology of mitochondrial gene expression in a range of cellular model systems.

Faithful DNA replication is essential for genome stability, yet replication forks face constant stress. The Bloom syndrome helicase (BLM) safeguards fork integrity, but excessive BLM activity can itself induce replication stress. We identify SLX4IP as a genome-wide regulator that restrains BLM to maintain replication fork stability. SLX4IP localizes broadly across chromatin with recruitment enhanced under replication stress. Loss of SLX4IP slows replication forks, remodels the replisome, and generates post-replicative single-stranded DNA gaps that are accompanied by elevated nuclear ADP ribose, reflecting compromised replication integrity. These defects are driven by dysregulated BLM activity, establishing SLX4IP as a negative regulator of BLM-dependent replication stress. At ALT telomeres, SLX4IP deficiency triggers ATR signaling, telomere fragility, and accumulation of ALT-associated PML bodies. Here, SLX4IP functions in parallel with FANCM to restrain BLM at ALT telomeres, with co-depletion of SLX4IP and FANCM causing synthetic lethality in ALT-positive cells, a phenotype fully rescued by BLM loss. Together, our results define SLX4IP as a critical genome-wide regulator of replication fork integrity and reveal SLX4IP as a potential vulnerability in ALT-positive cancers.