Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich elements in the 3' untranslated regions of certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knockout mice have demonstrated roles for three of the family members, TTP, ZFP36L1 (L1), and ZFP36L2 (L2), in inflammation, chorioallantoic fusion, and hematopoiesis, respectively. However, little is known about a recently-discovered TTP family member, ZFP36L3 (L3). Although L3 exhibits similar general biochemical functions to other members of the TTP family, initial studies of this family member revealed a number of unique characteristics.
First, L3 does not shuttle between the nucleus and cytoplasm like TTP, L1, and L2. Through studies of L3 deletion mutants, we determined that a nuclear localization signal that resides within the conserved tandem zinc finger domain was functional, although the C-terminal nuclear export sequence was non-functional. We then demonstrated that the unique repeat domain of L3 was responsible for the “full-time” cytoplasmic localization of the protein and was able to override the ability of the nuclear localization signal to direct transport into the nucleus.
In addition, L3 is specifically expressed in rodent yolk sac and placenta, while the other members of the TTP family exhibit relatively ubiquitous expression. We further examined the expression of L3 at both the RNA and protein level. Through northern and western blotting, we demonstrated the expression of L3 during mid-to-late gestation in mouse placenta. We also performed immunostaining of placental sections to demonstrate that this protein is exclusively expressed in the cytoplasm of the labyrinthine trophoblast cells and trophoblast giant cells of the placenta.
L3 most likely binds to and promotes the decay of a certain set of mRNA transcripts. Because of its specific sites of expression, we hypothesized that L3 may regulate the decay of a set of mRNAs that are important for the development or physiology of the placenta. We employed the ribonucleoprotein immunoprecipitation-microarray analysis of mouse placenta lysates to identify possible mRNA targets of L3. Our study identified approximately 400 transcripts that were enriched in immunoprecipitates using a highly specific L3 antibody. Some of these transcripts could be bound and downregulated by L3 in a physiological setting. Our top candidate transcript, based on relative enrichment and sequence analysis, was B-type natriuretic peptide, a hormone well-known for its role in cardiac physiology. We confirmed the expression of B-type natriuretic peptide in mouse placenta through northern blotting and in situ hybridization histochemistry. We also verified the ability of L3 to directly bind to and promote the degradation of this transcript in electrophoretic mobility shift assays and co-transfection assays, respectively.
Lastly, L3 demonstrates a unique migration characteristic in denaturing polyacrylamide gel electrophoresis as compared to TTP, L1, and L2. It migrates as two distinct species of Mr ~90,000 and ~100,000. We investigated the basis for this unusual migration in studies of deletion mutants and serine mutants. We found that both phosphorylation and the presence of the conserved C-terminus are required for the existence of the slower-migrating species. We then focused our study on phosphorylation of the C-terminus and discovered that the phosphorylation of Ser721 may play a role in creating the slower-migrating species. We also identified four other phosphorylated residues with mass spectrometry. Finally, we examined the effect of the C-terminus on the function of L3 and determined that this conserved region is not required for mRNA binding or to promote mRNA deadenylation or degradation in our assays.
The work described in this dissertation increases our understanding of this unique tristetraprolin family member, L3. Additional study of this protein is required to further elucidate its role in the physiology of rodent placenta, and to determine whether this role is subsumed by one of the other TTP family members in the placentas of other mammals.