Saba Valadkhan, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The long term goal of the proposed research is to understand, in detail, the role of spliceosomal snRNAs in catalysis of the splicing reaction. In the active spliceosome, U6 and U2 snRNAs form a base-paired complex that helps position the reactants of the first step of splicing for catalysis. Previously published work and preliminary studies show that an in vitro-assembled, base paired U6/U2 complex resembling the one forming in vivo can catalyze a reaction closely related to the first step of splicing. This finding will be pursued under three specific aims. 1) Characterization of the splicing-related catalytic activity of U6 and U2 snRNAs. To conclusively demonstrate that the reaction catalyzed by the in vitro-assembled U6/U2 complex is indeed identical to the first step of splicing, the chemistry of the reaction will be directly analyzed by nuclease digestion of site-specifically labeled products, followed by TLC analysis. In addition, by manipulating the structure of the U6/U2 complex, the basis for the selection of the scissile phosphate will be defined. 2) Determination of the structural organization of the catalytic U6/U2 complex. In vivo data has indicated the importance of the three-dimensional structure of the U6/U2 complex in spliceosomal catalysis. Using a battery of chemical probing reagents and crosslinking assays, the structural architecture of the in vitro- assembled U6/U2 and its interactions with the splicing substrates will be defined. 3) Functional analysis of the sequence elements required for catalysis. Mutational analysis of the snRNAs in the spliceosome has revealed a functionally crucial role for a number of nucleobases and backbone phosphates. The molecular basis of the function of these required elements will be defined using nucleotide analog interference mapping (NAIM). Using a similar approach, the functional significance of abundant post-transcriptional modifications in the U6 and U2 snRNAs will be defined. Almost all human pre-messenger RNAs undergo multiple splicing events, and alternative splicing is not only one of the most important means of regulation of gene expression, it is also largely responsible for generating proteomic diversity in eukaryotes. Disturbances in the pattern of pre-mRNA splicing have been linked to a broad spectrum of human diseases ranging from genetic and neurodegenerative diseases to malignancies. [unreadable] [unreadable] [unreadable]

Abstract The broad, long term goal of the proposed research is to identify new therapeutic targets against HIV through the study of long non-coding RNAs (lncRNAs), a novel class of genes that due to their relatively recent discovery, have remained largely unstudied. Despite the availability of highly effective anti-retroviral therapies, eradication of HIV has remained impossible. This is largely due to the inability of existing therapies to target the latent reservoir of transcriptionaly silent HIV proviruses. While our understanding of HIV latency is far from complete, the existing data point to an important role for transcriptional and epigenetic regulatory programs in HIV latency. Interestingly, emerging data indicate that lncRNAs play critical roles in both transcriptional and epigentic regulation, pointing to the potential of this class of cellular RNAs as therapeutic targets against HIV. As a first step toward addressing this exciting possibility, we have taken a high throughput sequencing approach to define the extent of involvement of lncRNAs in HIV life cycle, targeting the clinically crucial steps of early infection, latency and reactivation. Our preliminary studies have revealed that all the above-listed stages of HIV life cycle are associated with differential expression of a large number of lncRNAs. Some of the differentially expressed lncRNAs originated from genomic loci that were positioned in close proximity of protein coding genes with key roles in HIV biogenesis. Intriguingly, in some cases, the architecture of the locus pointed to the possibility of a regulatory interaction between the lncRNA and the protein-coding RNA. Validation studies on two such loci proved the strong regulatory impact of the lncRNAs on their protein-coding neighbors. These exciting results led to the hypothesis that such local, cis regulatory networks may play an important functional role in HIV biogenesis and lead to identification of novel therapeutic targets. We propose to address this hypothesis under three specific aims. In Aim 1, we will complement our existing high throughput sequencing approach by including additional stages of HIV life cycle, including earlier time points of primary infection, and early and intermediate stages of latency and proviral reactivation. These studies will not only reveal the true extent of involvement of lncRNAs in HIV life cycle, but also they are likely to provide additional examples of potential cis-regulatory networks operating in HIV biogenesis. In Aim 2, we will test the validity of the potential cis-regulatory loci identified in Aim 1 using RT-PCR-based methods after forcefully increasing or decreasing the level of the lncRNAs in each locus using shRNA or transgene-mediated overexpression methods. Finally, in Aim 3, we will determine whether the validated cis-regulatory relationships impact HIV biogenesis. The most promising candidates with the strongest impacts on HIV biogenesis will be selected for additional studies to further evaluate their potential as anti-HIV therapeutic targets. Taken together, this project will define the extent of involvement of lncRNAs in HIV life cycle, and will identify cis regulatory networks that may impact the replication, latency or reactivation of HIV.

Although metastasis is the most lethal characteristic of breast cancer (BC), our understanding of the molecular mechanisms that govern this event remains incomplete. Interestingly, many BCs disseminate long before their primary tumors become symptomatic. In fact, ~50% of women diagnosed with small tumors of the breast (4 mm) already harbor disseminated carcinoma cells in their bone marrow. Moreover, these micrometastases escape clinical detection by remaining latent for years before reemerging as incurable secondary tumors that are insensitive to chemotherapies that were originally effective against the primary tumor. A major barrier to eradicating BC reflects the paucity of knowledge related to how mammary tumors acquire metastatic and recurrent phenotypes, which underlies the inability of science and medicine to detect and treat latent micrometastases. These knowledge deficits are especially problematic for triple-negative breast cancers (TNBCs), which are highly aggressive and prone to rapid relapse; they also lack FDA-approved targeted therapies necessary to improve their dismal overall survival rates. Long noncoding RNAs (lncRNAs) have recently emerged as powerful global regulators of chromatin remodeling and gene expression in diverse physiological settings, including cell and tissue homeostasis, embryogenesis and development. Moreover, an ever expanding array of scientific evidence related to the pathophysiology of lncRNAs in human disease led us to postulate that developing and progressing TNBCs hijack the global chromatin reprogramming ability of lncRNAs, thereby eliciting emergence from metastatic latency and initiating lethal disease recurrence. Accordingly, we identified BORG (BMP/OP-Responsive Gene (BORG), as a powerful oncogenic lncRNA whose aberrant expression correlated with the acquisition of EMT (epithelial-mesenchymal transition) and metastatic phenotypes in (a) human and murine TNBCs cells; (b) human breast tumors and their corresponding CNS metastases; and (c) patient-derived xenograft (PDX) models of human BC as compared to normal breast epithelial cells. Additionally, BORG is sufficient in driving latent disseminated TNBCs cells to reactivate proliferative programs both in vitro and in vivo, events associated with epigenomic reprogramming operant in repressing cellular senescence programs, and in activating cell survival programs. Based on these and other preliminary findings, we hypothesize that BORG drives TNBC metastasis and recurrence by (i) remodeling the epigenome to reactivate proliferative programs that circumvent quiescence- and senescence-associated transcriptomes, and (ii) promoting the induction of survival signaling systems coupled to the acquisition of chemoresistant phenotypes. These hypotheses will be addressed by two Specific Aims. Aim 1 will determine the mechanisms whereby BORG:TRIM28 complexes form and drive TNBC metastasis and recurrence. We will identify the minimal BORG determinants necessary to bind TRIM28, as well as the domains in TRIM28 that bind BORG. Additionally, we will map the chromatin alterations provoked by BORG by performing TRIM28 and H3K4me1 ChIP-seq analyses in BORG-proficient and -deficient TNBCs, findings that will be validated in human breast cancer specimens. Aim 2 will determine the mechanisms whereby BORG:RPA1 complexes and NF-kB promote TNBC survival and chemoresistance. Similar to Aim 1, we will identify the minimal BORG sequences necessary to bind RPA1, and conversely, the regions in RPA1 that interact with BORG. The impact of preventing BORG:RPA1 complex formation on TNBC survival and chemoresistance will be determined, as will the role of ATM in regulating the interplay between TRIM28 and RPA1 when bound to BORG. Finally, the impact of these events in mediating TNBC resistance to cytotoxic chemotherapy will be assessed in vitro, and in preclinical therapy trials. Collectively, the findings obtained in this innovative application will provide novel molecular insights into how BORG drives TNBC to acquire metastatic, recurrent, and chemoresistant phenotypes.