Gene expression in mammalian systems is carefully tuned through transcriptional, post-transcriptional, and translation processes. This thesis focuses on the study of one post-translational chemical mark, S-palmitoylation, using chemical biology approaches, and the exploitation of translation regulation for therapeutic purposes, using synthetic biology approaches. S-Palmitoylation is a reversible post-translational lipid modification that has been observed on mitochondrial proteins, but both the regulation and functional consequences of mitochondrial S-palmitoylation are poorly understood. In this thesis, we first developed new members of the fluorogenic depalmitoylation probes (DPPs), DPP-5 and mitoDPPs. The DPP-5 features improved water solubility and incorporates the natural lipid substrate for enhanced S-depalmitoylases selectivity. The mitoDPPs preferentially localize to mitochondria to measure the level of mitochondrial activity of depalmitoylases in live cells. Using these probes, we discovered a new depalmitoylase, ABHD10, in mitochondria, and characterized this enzyme by biochemical and structural analyses. We showed that peroxiredoxin-5 (PRDX5), a key antioxidant protein, is a target of ABHD10, and discovered that ABHD10 regulates the S-palmitoylation status of the nucleophilic active site residue of PRDX5, providing a direct mechanistic connection between ABHD10-mediated S-depalmitoylation of PRDX5 and its antioxidant capacity.To address the therapeutic need for diseases caused by insufficient gene expression, we developed “translation-activating RNAs” (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. We show that we can construct taRNAs from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and demonstrate that taRNAs can activate gene expression from a suite of target mRNAs. We minimized the taRNA scaffold to 94 nucleotides, identified two translation initiation factor proteins responsible for taRNA activity, and validated the technology by amplifying SYNGAP1 expression, a target of haploinsufficiency disease, in patient-derived cells. Finally, we show that taRNAs can be delivered as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a compact and general nucleic acid-based technology to upregulate protein production from endogenous mRNAs, opening up new possibilities for therapeutic RNA research.