Browsing by Subject "RNA-binding proteins"
- Results Per Page
- Sort Options
Item Open Access Molecular and Evolutionary Analysis of RNA-Protein Interactions in Telomerase Regulation.(Non-coding RNA, 2024-06) Davis, Justin A; Chakrabarti, KausikTelomerase is an enzyme involved in the maintenance of telomeres. Telomere shortening due to the end-replication problem is a threat to the genome integrity of all eukaryotes. Telomerase inside cells depends on a myriad of protein-protein and RNA-protein interactions to properly assemble and regulate the function of the telomerase holoenzyme. These interactions are well studied in model eukaryotes, like humans, yeast, and the ciliated protozoan known as Tetrahymena thermophila. Emerging evidence also suggests that deep-branching eukaryotes, such as the parasitic protist Trypanosoma brucei require conserved and novel RNA-binding proteins for the assembly and function of their telomerase. In this review, we will discuss telomerase regulatory pathways in the context of telomerase-interacting proteins, with special attention paid to RNA-binding proteins. We will discuss these interactors on an evolutionary scale, from parasitic protists to humans, to provide a broader perspective on the extensive role that protein-protein and RNA-protein interactions play in regulating telomerase activity in eukaryotes.Item Embargo Small Molecule Modulation of RNA Tertiary Structures and RNA-Protein Interactions(2024) Martyr, JustinThe recently characterized therapeutic promise of noncoding RNAs has led to renewed vigor in the development of targeting strategies to manipulate their biological functions. While there have been strides in using primary sequence-based approaches to base-pair to specific regions of interest, small molecules offer unique promise in targeting RNA due to their favorable pharmacokinetics and synthetic accessibility. RNA-targeting small molecules have been used successfully in targeting more complex RNA secondary and tertiary structures, thereby altering function. However, due to the inherent flexibility and dynamics of RNA, there is a general lack of mechanistic understanding of how small molecules manipulate RNA conformational space to affect these functions. Among these biological functions is the ability to bind trans-acting factors, chief among them RNA-binding proteins, which can also modulate and shift the equilibrium of RNA conformations through their binding interactions. Therefore, developing a holistic understanding of how small molecules impact RNA structural space and thereby influence protein interactions can inform the next generation of RNA-targeting small molecule therapeutics.To this effect, we assessed an RNA-targeting focused library for interactions with an RNA triple helix at the 3'-end of the cancer-associated long noncoding RNA MALAT1. We evaluated these interactions using affinity-based assays as well as applying an in vitro RT-qPCR assay to measure small molecule impacts to structural stability. In an effort to explore the cheminformatic properties driving these interactions, we developed quantitative structure-activity relationship models for small molecule binding and stability shifts. We further employed these models to predict and select novel molecules to test the model’s predictive power for functional outputs. This study demonstrates the feasibility of using these models to inform synthetic selection with predictive function, as well as providing high affinity probes for future cellular targeting of MALAT1. To evaluate RNA-binding proteins and how they interact with different RNA structures, we applied stability-based mass spectrometry proteomics to evaluate changes in protein stability upon RNA addition. Using the aforementioned noncoding RNA triple helix, a viral RNA stem-loop, and single stranded RNA sequence from the untranslated region of a coding mRNA, we used thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX) to interrogate RNA-protein interactions on the proteomic scale. We found that these methods, which require heat and denaturant to shift the protein folding equilibrium, can shift the equilibrium of the RNA conformational space, leading to the identification of RNA-binding protein hits that did not arise in more common pull-down methods. These tools provide novel avenues to interrogate RNA-binding proteins in a greater RNA dynamic context and could also be applied to the study of RNA-small molecule targeting to evaluate changes in protein stability through small molecule-induced RNA conformational shifts. We holistically evaluated small molecule-induced conformational change and influence on protein binding through the scope of RNA G-quadruplexes. We investigated the targetability and modulation of RNA G-quadruplexes using our RNA-targeting small molecule focused library, as well as measured how these molecules impact G-quadruplex-protein interactions. By identifying small molecules which could either stabilize or unfold a model RNA G-quadruplex, we then applied the SPROX workflow to identify how proteins shifted in stability in the presence of the G-quadruplex and when small molecules were added. With the application of mass spectrometry, we compiled a comprehensive list of proteins which change in stability upon small molecule addition, suggesting impacts to these RNA-protein interactions in a small molecule dependent manner. This work is the first to highlight this dynamic relationship between small molecule, protein, and RNA G-quadruplex conformational space on the proteomic scale. The work discussed herein provided insights into small molecule properties which lead to differential RNA-small molecule recognition and structural modulation. Additionally, this work highlights the use of stability-based mass spectrometry tools to assay dynamic RNA-protein interactions. Taken together, these advances will boost future RNA-targeting efforts with tools to investigate how small molecules both modulate RNA conformational dynamics and further manipulate RNA-protein interactions.
Item Embargo ZFP36L2 in Development and Adulthood: A Critical Regulator of Hematopoietic Stem Cell Homeostasis(2023) Huang, RuiThe 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.