The RNA binding protein tristetraprolin (TTP) is an mRNA destabilizing factor that regulates the stability of transcripts involved in promoting inflammation. TTP regulates mRNA stability through recruitment of decay factors involved in deadenylation, decapping, exonucleolytic decay, and translation repression. TTP activity is regulated by post-translational modifications with nearly 30 reported sites of phosphorylation; however, the functional effects of only two sites are well characterized. Previous work revealed the conserved TTP CNOT1 Interaction Motif (CIM) is an important structural element that promotes the association with the major deadenylase complex, CCR4-NOT. In the work described in Chapter 2, I studied how the TTP CIM promotes mRNA decay cooperatively with other regions of the TTP protein. Using tethered decay assays, I find that mutation of conserved tryptophan residues, which associate with the CCR4-NOT subunit CNOT9, in addition to removing the CIM causes stabilization of target mRNA. Pull-down assays of TTP mutants reveal that CCR4-NOT recruitment is facilitated in a cooperative manner by the TTP-CIM and tryptophan residues. Furthermore, I find, contrasting previous reports, that the p38 MAPK pathway does not target a conserved serine residue within the TTP-CIM and that the CIM remains active in conditions where TTP is targeted by the downstream p38 MAPK kinase, MK2. These results suggest that efficient regulation of TTP requires multiple signaling pathways. Difficulties examining TTP under native conditions has been a limitation of previous studies. In the work described in Chapter 3, to facilitate studies of TTP in a more endogenous setting, I generated TTP-/- RAW 264.7 mouse macrophage cells using a CRISPR-Cas9 targeting approach. These were used to generate lentiviral add-back cells expressing TTP from the endogenous TTP promoter. Unfortunately, exogenous TTP expression was unable to reach the levels seen from the endogenous locus in RAW 264.7 cells suggesting that additional enhancer element(s) acting on the TTP locus are necessary to produce an efficient TTP-expression response. Additional repair template-mediated CRISPR-Cas9 gene editing techniques using selectable markers are also reported here as a promising tool to generate endogenous TTP mutants in RAW 264.7 macrophage cells.