Noncanonical role of ALAS1 as a heme-independent inhibitor of small RNA–mediated silencing [Ago2-CLEAR-CLIP]

RNA interference (RNAi) is a fundamental strategy for gene silencing via small interfering RNAs (siRNAs) that program Argonaute (Ago) effectors, and is related to endogenous silencing via microRNAs (miRNAs). Although most efforts focus on factors that are required for miRNA/RNAi function, biological systems are typically subject to repression. Using molecular genetic approaches, we uncover ALAS1, the first enzyme in the heme biosynthesis pathway, as a repressor of miRNA accumulation in cells and in mice. This was non-intuitive, since heme is known to play a positive role as a cofactor for the nuclear miRNA processing machinery. Instead, we show that ALAS1 (but not other core heme biosynthesis factors) limits RISC assembly and activity. Notably, ALAS1 is the target of one the few current FDA-approved siRNA drugs (Givosiran). Our data have implications for side effects of Givosiran, but also for RNAi therapies in general. We provide evidence that depletion of ALAS1 enhances siRNA-mediated knockdown, suggesting that Givosiran is a viable RNAi adjuvant. To investigate functional effects on miRNA-mediated targeting upon loss of ALAS1, we performed Ago2 CLEAR-CLIP