Regulating the precise rate of protein production from each protein-coding gene is a fundamental process of all cellular life. While transcriptional regulation plays a large role in determining final protein levels, post-transcriptional events can also make substantial contributions. In mammals, the majority of the cis-regulatory information that controls post-transcriptional events is located within a transcript’s 3′ untranslated region (3′ UTR). The cis-regulatory sequence elements (cis-elements) found within 3′ UTRs are bound by trans-acting factors, mainly RNA binding proteins and non-coding RNAs, which in turn interact with the core decay and translation machineries to modulate mRNA decay or protein synthesis rates. Though a large number of cis-elements have been identified, many questions remain about their distribution and interactions. In addition, the contribution of parameters whose function is independent of their sequence, such as the length of the 3′ UTR, to gene regulation is poorly understood. Numerous studies have established that typical 3′ UTRs contain multiple discrete cis-elements, yet the typical density of elements within 3′ UTRs is unclear. Moreover, examples exist describing consequential interactions between cis-elements, either cooperative or inhibitory. However, the extent to which such interactions are a general paradigm for cis-elements remains to be determined. By performing a systematic study of the regulatory sequences within two conserved mammalian 3′ UTRs, those of Hmga2 and PIM1, I determined that both 3′ UTRs contain a high density of cis-elements (at minimum 6 and 12 per kb, respectively) spread across the entire 3′UTR. Importantly, the vast majority of the cis-elements function independently of neighboring elements. Additionally, despite the overall repressive effect of the 3′ UTRs, I found that many regulatory cis-elements enhance gene expression, rather than repressing it. I hypothesize that the enhancing cis-elements counteract a repressive effect of 3′ UTR length. In a second study, I explored the effect of 3′ UTR length on gene expression using, as 3′UTR mimics, randomly-generated, nucleotide-composition matched, sequences of varying lengths. Long 3′ UTRs have previously been identified as targets of an mRNA surveillance mechanism called nonsense-mediated decay (NMD). In this study, I discovered a novel role for 3′ UTR length in triggering an NMD-independent decay pathway in human cell lines. Reporter transcripts with random 3′ UTR mimics as short as 400 nucleotides were repressed by this pathway, with the repression growing stronger with increasing length. While the mechanism of this novel pathway remains to be elucidated, I have determined that it affects the decay rate of mature mRNAs in a deadenylation-independent manner. Overall, by determining the density and extent of interactions of cis-element within example mammalian 3′ UTRs and by identifying a novel role for 3′ UTR length in regulating gene expression, this work furthers our understanding of fundamental aspects of 3′ UTR-mediated gene regulation.