Mapping Functional Regions within the N-Terminus of Human Pumilio Protein
One of the key steps for gene regulation occurs at the mRNA level. RNA-binding proteins, such as the human Pumilio (PUM) proteins, can bind and regulate mRNA. Pumilio binds a specific consensus sequence within the 3′-untranslated region of mRNA and then promotes mRNA deadenylation and degradation through interaction with the CCR4-NOT (CNOT) complex. Pumilio structure for binding mRNA has been well characterized, but the protein-protein contacts with CNOT remain to be determined. Here we analyzed the function of two highly conserved regions within the N-terminus of human Pumilio proteins termed Pumilio Conserved Motif A (PCMa) and Pumilio Conserved Motif B (PCMb). Based on previous studies in Drosophila Pumilio, it was hypothesized that these two regions were autoregulatory. To determine the function of these regions we used a luciferase-based reporter assay system in HCT116 cell lines. Our results demonstrate that PCMa and PCMb in human PUMs are not autoregulatory in nature but contain repression activity on their own. This indicates two new regions of importance for PUM repression function and regulation of mRNA. The repression activity of these regions may lead to a novel mechanism for repression by PUMs and its regulation of mRNA transcripts relating to development and cell cycle control.