Browsing by Subject "lncRNA"

Osteoarthritis (OA) is one of the leading causes of disability in the United States, afflicting over 27 million Americans and imposing an economic burden of more than $128 billion each year (1, 2). OA is characterized by progressive degeneration of articular cartilage together with sub-chondral bone remodeling and synovial joint inflammation. Currently, OA treatments are limited, and inadequate to restore the joint to its full functionality.

Over the years, progresses have been made to create biologic cartilage substitutes. However, the repair of degenerated cartilage remains challenging due to its complex architecture and limited capability to integrate with surrounding tissues. Hence, there exists a need to create not only functional chondral constructs, but functional osteochondral constructs, which could potentially enhance affixing properties of cartilage implants utilizing the underlying bone. Furthermore, the molecular mechanisms driving chondrogenesis are still not fully understood. Therefore, detailed transcriptomic profiling would bring forth the progression of not only genes, but gene entities and networks that orchestrate this process.

Bone-marrow derived mesenchymal stem cells (MSCs) are routinely utilized to create cartilage constructs in vitro for the study of chondrogenesis. In this work, we set out to examine the underlying mechanisms of these cells, as well as the intricate gene correlation networks over the time course of lineage development. We first asked the question of how transforming growth factors are determining MSC differentiation, and subsequently utilized genetic engineering to manipulate this pathway to create an osteochondral construct. Next, we performed high-throughput next-generation sequencing to profile the dynamics of MSC transcriptomes over the time course of chondrogenesis. Bioinformatics analyses of these big data have yielded a multitude of information: the chondrogenic functional module, the associated gene ontologies, and finally the elucidation of GRASLND and its crucial function in chondrogenesis. We extended our results with a detailed molecular characterization of GRASLND and its underlying mechanisms. We showed that GRASLND could enhance chondrogenesis, and thus proposed its therapeutic use in cartilage tissue engineering as well as in the treatment of OA.

The post translational modification (PTM) of histone proteins is a highly dynamic process that is utilized in the control of gene transcription. This epigenetic process involves enzymatic ‘writers’ and ‘erasers’ which place or remove chemical modifications to the unstructured tails of histone proteins which protrude out from the nucleosomal core. In a highly dynamic manner, each PTM is spatiotemporally regulated and combinations of PTMs at a gene promotor or enhancer region leads to transcriptional enhancement or repression. The gene targets as well as selectivity and specificity of epigenetic enzymes is regulated by the multimeric complexes each enzyme is co-opted. Each complex contains a unique set of coregulatory proteins with RNA and DNA binding domains and PTM ‘reader’ domains to direct the catalytic machinery to a specific subset of genes. The coregulatory proteins also affect the specificity and selectivity of the enzyme through mechanisms which are only beginning to be explored.

Our interest is in elucidating the role of coregulatory proteins and lncRNA with respect to lysine-specific demethylase 1 (LSD1/KDM1A). A flavin-dependent mono-and di-demethylase of H3K4me1/2 and H3K9me1/2, KDM1A has been implicated in many different multimeric enzymatic complexes which, in some cases such as the REST and NuRD complexes, function on opposing pathways. This disparity in the downstream outcome being coordinated by the same enzyme highlights the need to understand not only epigenetic enzymes, but to consider the complexes as a whole towards therapeutic targeting.

The specific aims of my thesis were to (a) interrogate the role of individual and multiple coregulatory partners in enzyme selectivity and specificity (b) establish tools to study the mechanisms of biochemical and biophysical of protein:protein and protein:lncRNA interactions and (c) elucidate key characteristics of protein:protein and protein:lncRNA interfaces towards targeted disruption. To this end, I have utilized cloning and mutagenesis methods to heterologously express and purify coregulatory partners of KDM1A in E. coli. I chose coregulatory partners found in a common catalytic core as well as several additional coregulatory proteins from a stable KDM1A-containing 5-mer complex. I have produced multiple constructs for four of these proteins to allow for multiple affinity purification routes as well as for future binding studies. I have further expressed each of these constructs and have made significant efforts towards the purification of each construct based on solubility.

I furthermore established HDX-MS and SELEX protocol in our lab as tools to allow us to explore the dynamics of these epigenetic interactions. I further demonstrated and confirmed that there is no hotspot along the binding interface between KDM1A and CoREST, but that CoREST stabilizes the apical end of the KDM1A tower domain via HDX-MS with the highest change in deuterium uptake, over 20%, long KDM1A TαA residues 440-451.

I also made significant efforts towards elucidating the interaction between KDM1A and HOTAIR. Firstly, I established an RNA radiolabeled EMSA assay for the lab which allowed us to test the binding of HOTAIR to KDM1A. With this assay, we saw that CoREST286-482, specifically the linker region (residues 293-380), must be bound to KDM1A for HOTAIR to bind and that the dissociation constant was unchanged at 1.710.38 µM and 1.29±0.34 µM, respectively. Further, I confirmed that the first 320 nt of domain 4 of HOTAIR (nt 1500-1820) contain the critical binding and that the dissociation constant was slightly higher at 2.97±0.96 µM.

I have also optimized SHAPE-MaP and crosslinking strategies to explore the binding interface between KDM1A:CoREST286-482 and lncRNA. I determined that there were 83 nt that displayed at least a 1.5-fold change in SHAPE reactivity of HOTAIR D4 due to the presence of KDM1A:CoREST286-482. I also utilized a free-energy based secondary structure model to establish a secondary structure for HOTAIR D4 based on my SHAPE-MaP data. I noted that 44% of the significant nt were confined to a stretch of RNA (nt 1538-1610, 1779-1844) that is predominantly dsRNA. Further usage of photochemical crosslinking strategies revealed a propensity for G:C paired nt to be crosslinked to KDM1A:CoREST286-482. A similar nt sequence around these paired nt suggests a binding motif. The implications of these results is discussed herein.

The discovery of the many active roles of RNA in diseased pathways propelled the current ‘RNA Renaissance’. As a result, RNA is no longer regarded as a passive intermediate between DNA and proteins, but as a key player in human disease and an attractive therapeutic target. Small molecules are uniquely poised for RNA-targeting due to their potential for favorable bioavailability, cell permeability, and spatio-temporal control, overcoming the current limitations of gene editing and sequence-based technologies. Nevertheless, questions regarding favorable properties that render small molecules specific for RNA and, ideally, selective for one RNA target over others are far from being answered. Thus, to elucidate possible trends that can expedite future targeting efforts we leveraged both synthetic and commercially available approaches in targeting disease relevant structures in mammalian and viral RNAs. We designed and synthesized a focused library and evaluated it using a holistic approach against the RNA triple helix motif present at the 3’-end of the mammalian oncogenic long non-coding RNA MALAT1. The development of triple helix-focused assays enabled us to assess small molecules’ affinity and their effect on the triplex structural stability and enzymatic digestion. This study resulted in the first example of synthetically tuned small molecules that can bi-directionally modulate the susceptibility of the triplex to enzymatic degradation. In a subsequent study we leveraged the well-established relationship between the structural stability of the MALAT1 triple helix and its refractivity to degradation as a case-study to develop a high-throughput platform that directly reports on the effects of mutations or small molecules on the stability of the triple helix. Optimization and application of this platform to RNA G-quadruplexes and pseudoknots corroborates the potential of this assay to bridge the current gap between affinity-based assays and cell-based activity. With the emergence of SARS-CoV-2, we focused our efforts in identifying potential RNA targets within the viral genome that would be amenable to small molecule targeting with the goal of developing new antiviral agents. Specifically, we first focused on conserved RNA structures at the 5’-end of the viral genome and utilized an in-house focused library based on the known RNA-binding scaffold amiloride. Through evaluation of small molecules leads using a plethora of in vitro and in cellulo assays we identified three small molecule leads that inhibited viral replication by targeting structures at the 5’-end of the genome. This study represented the first example of RNA-targeted SARS-CoV-2 antivirals. We then focused on an RNA structure present at the interface of two overlapping open reading frames and responsible for programmed ribosomal frameshifting, and essential process in coronavirus lifecycle. Screening of an RNA-biased commercially available library curated by our laboratory resulted in the identification of a small molecule selective for the CoV-2 pseudoknot over other disease-relevant triple helices. The work presented herein provides essential insights into RNA:small molecules molecular recognition events and provides tools and workflows that will significantly expedite future targeting efforts against the RNA structures presented in this work and others yet to be identified or explored.

The noncoding RNA (ncRNA) revolution has revealed myriad RNA species that play critical roles at all stages of life, including embryogenesis and disease progression. For example, three long ncRNA (lncRNA), Hox Transcript Antisense Intergenic RNA (HOTAIR; ~2.5 kb), Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1; ~6.7 kb) and Second Chromosome Locus Associated with Prostate-1 (SChLAP1; ~1.5 kb), are basally expressed in normal prostate tissue but are dysregulated in prostate cancer. HOTAIR scaffolds the PRC2 and LSD1 protein complexes to selectively methylate and demethylate, respectively, histone proteins, thereby regulating downstream gene expression. MALAT1 acts in trans at nuclear speckles during mRNA post-transcriptional processing while SChLAP1 acts in cis to influence oncogenic gene expression. Initial work on HOTAIR developed technical skills that were then applied to the lncRNAs MALAT and SChLAP1 to learn more about their role in prostate cancer. There is compelling evidence that the 3′–end of MALAT1 is a triple helix structure that acts as a molecular knot, driving transcript accumulation in cancer cells and furthering their metastatic potential, but we currently lack any biophysical data detailing the relationship between SChLAP1 structure and function. In general, the relationship among lncRNA structure, dynamics, and function is not well understood; for example, even with high-resolution structures of the MALAT1 triple helix, questions remain regarding the role of intrinsic dynamics in transcript stability or protein binding. As lncRNA represent an underexplored therapeutic avenue, this work aims to investigate the role of lncRNA structure and dynamics in driving prostate cancer metastasis. This work uses biophysical and biochemical methods including chemical probing, NMR, SAXS, and native gels to study the two lncRNA MALAT1 and SChLAP1 and learn more about their respective structure-function relationships. From this work, we have found a discrete structure within the lncRNA SChLAP1 that is highly structured and implicated in driving metastasis via protein recognition. Additionally, our preliminary studies regarding MALAT1 support the presence of non-triplex states that require further characterization. Overall, this work supports a deeper understanding of lncRNA structure as it relates to their function in cancer and provides examples for the biophysical analysis of large and/or structurally complex RNA.