No abstract

Suppression of premature termination codons (PTC) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two novel and potent readthrough promoters - NVS1.1 and NVS2.1 - that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, activating a novel branch of the ribosome-associated quality control (RQC) network that involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.

No abstract

Translation is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for many decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows the preparation of cytoplasmic extracts from human cells that efficiently translate mRNAs in a capped and IRES-mediated way. We optimized lysate preparation and in vitro translation conditions and show that dual centrifugation allows the production of human lysates under detergent-free conditions and is ideally suited to produce ample amounts of human translation-competent lysates.