Browsing by Subject "Tristetraprolin"

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.

Posttranscriptional gene expression is controlled and coordinated by RNA binding proteins (RBPs), many of which recognize specific RNAs through cis-regulatory RNA elements. One of the most highly studied classes of cis-regulatory RNA elements is the AU-rich elements (AREs). AREs are bound by a class of RBPs called ARE binding proteins (ARE-BPs), of which there are over a dozen in humans including HuR, tristetraprolin (TTP) and butyrate response factors 1 and 2 (BRF1 and BRF2). TTP, BRF1 and BRF2 belong to a family of tandem C3H zinc finger proteins that destabilize ARE-containing mRNAs. HuR acts to enhance the stability and translation of ARE-containing mRNAs, a function that is rare among ARE-BPs. While each of these ARE-BPs regulates the expression of ARE-containing mRNAs, some ARE-BPs themselves are also encoded by ARE-containing mRNAs, raising the possibility that each of these ARE-BPs may regulate one another's expression. In order to determine how these ARE-BPs influence each others expression and how this affects the regulation of global gene expression programs we have focused on three different aspects of these ARE-BP networks: control, response to stimuli, and global effects.

To address of network control of ARE-BPs we have focused on how HuR regulates a network of mRNAs including TTP, BRF1 and HuR's own mRNA. We demonstrate that HuR can bind to TTP's, BRF1's and its own mRNA. Furthermore, by employing overexpression and siRNA knockdown approaches we demonstrate that these mRNAs and their corresponding 3'UTR luciferase reporters are resilient to fluctuations in HuR levels and that the degree of this resiliency is cell type and condition specific.

To address the temporal responses within an ARE-BP network we focused on how each of the members of the TTP family of ARE-BPs reacts following the induction of the other family members by using epidermal growth factor (EGF) stimulation. Here we show that induction of TTP family member mRNAs during EGF stimulation is partially attributable to changes in mRNA stability. Furthermore, we also show that TTP and BRF1 are able to bind each of the TTP family member mRNAs and subsequently affect their expression by altering their mRNA degradation rates. In addition, we demonstrate that the unique temporal induction patterns of the TTP family member RBPs is correlated with the EGF stimulated induction of TTP-bound mRNAs, suggesting that a network comprised of TTP family members is able to influence the timing of complex gene expression patterns.

Finally, to address the influence of these networks on regulation of global gene expression programs we have focused on how HuR recognizes AREs and whether it can globally recognize multiple classes of ARE-containing mRNAs, including the canonical class of AREs recognized by the TTP family members. To investigate how the three RNA recognition motifs (RRMs) of HuR contribute to ARE recognition we generated a series of RRM point mutants and test their ability to disrupt RNA recognition of each of the RRMs. To identify different classes of ARE-containing mRNAs we examined these mutants with a global RNA binding site detection method called photoactivatable ribonucleoside crosslinking immunoprecipitation (PAR-CLIP). Together these techniques suggest that the RRMs of HuR cooperate to recognize mRNA targets and that HuR's ability to bind RNA is coupled to the cellular distribution of HuR, and thus, are important in its role for regulating expression of bound mRNAs.

Together these studies indicate that ARE-BP posttranscriptional networks are highly interconnected and display complex regulatory interactions depending on cell type and stimuli. Furthermore, these networks can create complex behaviors such as timing of expression events or resiliency to fluctuations in protein levels. Finally, the components of these ARE-BP networks target partially overlapping sets of mRNAs to impact global gene expression patterns that ultimately coordinate the cellular responses to external stimuli.

The tristetraprolin (TTP) protein family of RNA-binding proteins contains three widely expressed mammalian protein members: TTP (ZFP36), ZFP36L1, and ZFP36L2, all of which can regulate gene expression by binding to specific AU-rich sequences located in the 3'-untranslated regions (3’-UTR) of mRNAs and accelerating their decay. Unique among the three, ZFP36L2 plays a pivotal role in maintaining hematopoietic stem cells (HSC) during development. ZFP36L2-deficient mice exhibit severely impaired definitive hematopoiesis and die approximately two weeks after birth due to severe anemia, thrombocytopenia, and internal hemorrhage. Recent single-cell RNA sequencing (scRNA-seq) studies have demonstrated widespread Zfp36l2 expression in HSC and the hematopoietic system during both development and adulthood.

Despite the recognized importance of ZFP36L2 in maintaining HSC, there are still numerous aspects of its role that are not fully understood, hindering our understanding of hematopoietic regulation. While prior studies have provided valuable insights into the physiological function of ZFP36L2, its molecular mechanisms in HSC development and hematopoietic system maintenance remain poorly defined. To decipher its involvement in hematopoiesis, it is crucial to identify the mRNA targets and pathways regulated by ZFP36L2 and determine whether this regulation is intrinsic to HSC. Moreover, the premature death of ZFP36L2-deficient mice makes it unclear to what extent this protein governs adult HSC function. Lastly, considering the pivotal position of HSC in the hematopoietic hierarchy, it is important to investigate how ZFP36L2's activity in HSC affects the subsequent differentiation of hematopoietic lineages. Clarifying this relationship could yield valuable insights into the post-transcriptional mechanisms that govern HSC biology and potentially lead to the identification of new therapeutic targets for hematological disorders.

To address the knowledge gap, we employed a detailed analysis combining flow cytometry and scRNA-seq to examine HSC and hematopoietic progenitor cells (HSPC) at several critical developmental stages. Our studies revealed that the absence of ZFP36L2 resulted in significant reductions in both HSC and immature progenitors during mouse development, primarily due to HSC-autonomous dysregulation. In addition, scRNA-seq analysis of HSC and progenitors revealed that ZFP36L2 deficiency caused abnormal upregulation of transcripts related to cell cycle regulation and lymphoid specification, leading to aberrant cell cycle progression and premature lymphoid lineage commitment. This ultimately resulted in cellular damage and HSC exhaustion at birth. These findings demonstrate that ZFP36L2 is essential for maintaining the homeostasis of HSC, and emphasizes the significance of restraining lineage commitment and excessive self-renewal during HSC development.

In a related study, we investigated possible functional overlap of ZFP36L2 and TTP. We developed mice (L2KO/TTP∆ARE) that lacked Zfp36l2 but modestly overexpressed TTP throughout the body. L2KO/TTP∆ARE mice not only survived but also exhibited normal peripheral blood counts, except for residual moderate thrombocytopenia. We took advantage of this rescued ZFP36L2-deficient model and investigated the role of ZFP36L2 in adult hematopoiesis. We discovered that megakaryocyte (MK) progenitors and MK-biased HSC were decreased in bone marrow from L2KO/TTP∆ARE mice and exhibited enriched erythroid and decreased MK gene signatures. In addition, L2KO/TTP∆ARE HSC failed to reconstitute hematopoiesis upon non-competitive transplantation, and showed molecular features of stress and reduced cycling. Thus, TTP can assume some functions of ZFP36L2 in a genetic dose-dependent manner, but ZFP36L2 may be specifically required for the maintenance of megakaryopoiesis and HSC function.In summary, our studies provide novel insights into the essential role of ZFP36L2 in the maintenance of HSC throughout both developmental stages and adulthood. We demonstrate that ZFP36L2 is essential for restraining abnormal cell cycling and lymphoid commitment during HSC development, thereby ensuring proper HSC maintenance. Interestingly, our results also suggest that TTP may partially compensate for ZFP36L2 deficiency during the development of the hematopoietic system, but that ZFP36L2 may have specific functions in maintaining megakaryopoiesis and HSC function in adulthood. Overall, our research provides a strong foundation for future studies aimed at elucidating the underlying mechanisms that govern ZFP36L2's function in hematopoiesis, further advancing our understanding of the intricate regulatory paradigm of HSC biology.

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.