Browsing by Subject "Argonaute Proteins"
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Item Building the Human Argonaute 2 Interaction Network(2015-07-27) Kalantari, Roya; Mendelson, Carole R.; Corey, David R.; Liu, Qinghua; Olson, Eric N.; Yu, HongtaoRNA interference (RNAi) is a system that has been largely studied and defined by its ability to affect gene expression and translation in the cytoplasm. However, Argonaute (AGO) proteins, which are the major catalytic component of the RNA-induced silencing complex (RISC), have been found to be involved in nuclear roles outside of the canonical RNAi pathway. Within non-mammalian systems such as yeast and plants, AGOs have been shown to be involved in functions such as DNA methylation and heterochromatin formation. My laboratory has utilized systems involving small RNAs to target nuclear events such as transcription and splicing in human cells. In the case of transcription, we have shown that small RNAs are capable of targeting long noncoding RNAs (lncRNAs) along both the promoter and past the 3’ end of genes in order to control gene expression. We have also demonstrated that targeting of small RNAs to introns and exons of pre-mRNA can robustly alter the splicing pattern. Within these systems, we have found that AGO proteins are recruited by the small RNAs to the nuclear target. However, the protein-protein interactions and mechanisms involved remain unclear. Identification and understanding of the interactions of AGO proteins in the nucleus is essential for comprehension of the mechanisms by which these proteins act. I have studied AGO2 interactions through immunoprecipitation and a novel semi-quantitative mass spectrometry technique known as the Normalized Spectral Index Method (SINQ). Stringent screening of mass spectrometry results identified interactions with the TRNC6 proteins, and AGO3. Most cytoplasmic interacting partners were also partners for AGO2 in the nucleus, however interactions with the loading complex was impaired although it is present in nuclei. These data demonstrate that the core RNAi machinery is largely conserved between cytoplasm and nucleus. This work opens new avenues for the utilization of small RNAs in gene regulation both in the laboratory and in the clinic.Item Human GW182 Paralogs Are the Central Organizers for RNA-Mediate Control of Transcription(2017-07-28) Hicks, Jessica Anne; Conrad, Nicholas; Mangelsdorf, David J.; Brugarolas, James B.; Kraus, W. Lee; Corey, David R.RNA interference (RNAi) is an endogenous mechanism for regulating gene expression that can be manipulated for experimental or therapeutic purposes to knockdown protein expression. In the mammalian cell cytoplasm, small RNAs direct RNAi proteins to inhibit translation by regulating mRNA stability. In non-mammalian cell nuclei, RNAi proteins control gene transcription. Recently, in mammalian cell nuclei, the RNAi proteins were shown to control transcription and splicing. Despite what is known about RNAi factors in non-mammalian cell nuclei, the mechanisms of mammalian RNA-mediated nuclear regulation are not well understood and remain controversial, hindering the effective application of nuclear RNAi and blinding investigation of its natural regulatory roles. Argonaute 2 (AGO2) and TNRC6A (GW182) are the core proteins of RNAi. To better understand RNAi-mediated nuclear gene regulation, my goal was to use semi-quantitative (SINQ) mass spectrometry analyses on purified protein complexes to build a protein interaction network of nuclear AGO2 and TNRC6A. The stable interactions of AGO2 detected with this protocol were the TNRC6A, B, and C paralogs. While this did not reveal many proteins, it did provide a new direction to take with mass spectrometry analyses. Since the TNRC6 paralogs are stable interacting partners, analysis on those protein complexes were performed to reveal a new shell of nuclear RNAi interactions. Mass spectrometry of TNRC6A protein complexes revealed these proteins are central to forming interactions between the RNAi machinery and many proteins involved in transcriptional regulation. TNRC6A interactions include the AGO proteins, CCR4-NOT complex, histone modifiers, and the mediator complex. In addition, novel interactions with four DNA damage repair proteins were identified, providing another direction for future investigation. Functional analysis revealed that TNRC6, AGO2, NAT10 (histone acetylation), WDR5 (H3K4me3), and MED14 (Mediator) proteins are involved in RNA-mediated COX-2 transcriptional activation. Taken together, the mass spectrometry and functional experiments provide evidence that these RNA-AGO-TNRC6 complexes act globally in cell nuclei to regulate transcription. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. The significance of my data is that it can lead to new advances in RNAi capabilities beyond canonical applications.Item Regulatory Mechanisms of the MicroRNA Pathway(2017-10-25) Golden, Ryan Joshua; Mendelson, Carole R.; Mendell, Joshua T.; Russell, David W.; Liu, QinghuaMicroRNAs (miRNAs) associate with members of the Argonaute protein family and downregulate partially complementary messenger RNAs (mRNAs) (1). miRNA activity is tightly regulated during development and in normal physiologic settings, while gain or loss of these control mechanisms can contribute to disease (2-4). To identify new mechanisms that regulate the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening (5, 6) coupled with a fluorescent miRNA pathway reporter. These experiments revealed an unanticipated role for the ANKRD52-PPP6C serine/threonine phosphatase complex as a critical regulator of miRNA activity in human cells. Loss of this complex significantly impaired global miRNA function. Genetic and biochemical studies revealed that phosphorylation of Argonaute2 (AGO2) on a set of highly conserved serine residues, S824-S834, blocks target mRNA engagement. Constitutive activity of the ANKRD52-PPP6C complex is necessary to remove these inhibitory phosphates and thereby allow miRNA-mediated silencing. A genome-wide CRISPR-Cas9 suppressor screen performed in ANKRD52-/- cells identified CSNK1A1 as the inhibitory AGO2 kinase that phosphorylates these sites. Together, these findings reveal a previously uncharacterized AGO2 phosphorylation cycle, uncovering a major mechanism through which the miRNA pathway is regulated and highlighting the power of iterative CRISPR-Cas9 screening for the dissection of biological pathways directly in human cells.Item The Role of TNRC6 in RNA Interference(2022-05) Johnson, Samantha Tori; Liu, Yi; Conrad, Nicholas; Kraus, W. Lee; Corey, David R.Small RNAs can influence translation, splicing, transcriptional activation, and transcriptional repression, through the RNA interference (RNAi) pathway. TNRC6, also known as GW182, and Argonaute (AGO) are the core proteins of RNAi. TNRC6 is a scaffolding protein that associates with AGO and bridges its interactions with other proteins to control gene expression in many different processes. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C. These paralogs share approximately 40% amino acid sequence identity. Whether the paralogs have unique or redundant functions is unclear. Much is known about the mechanisms of cytoplasmic RNAi but the world of nuclear RNAi remains murky. We understand that RNAi factors are present and active in the nucleus, but endogenous nuclear RNAi functions are unknown. I have used a suite of gene knockout cell lines for the TNRC6 paralogs to learn more about TNRC6 paralogs and their roles in RNAi. I examined if TNRC6 paralogs were required in several functions of small duplex RNA-mediated control of gene expression, including translational silencing by miRNAs, translational silencing by siRNAs, and transcriptional activation. On a global scale, I used high-throughput RNA sequencing, mass spectrometry, and enhanced crosslinking immunoprecipitation (eCLIP) to gather definitive answers about the TNRC6 paralogs, their roles in the cell, and their relation to AGO knockout cell lines. I found that that despite less than 40% sequence identity, the TNRC6 paralogs are functionally redundant and can replace one another for core RNAi functions. Each subsequent TNRC6 paralog knockout caused more gene changes and few genes overlapped between the single TNRC6 paralog knockouts. Changes in levels of gene expression in TNRC6 knockout cell lines are well-correlated with those observed in AGO knockout cell lines, emphasizing the important regulatory function of the partnership between AGO and TNRC6 in endogenous RNAi. Further, I found TNRC6 plays a role in splicing changes initiated by small RNA binding to intronic regions. Taken together these data further define the roles of the TNRC6 paralogs as part of the RNA interference machinery. TNRC6 is a close protein partner of AGO and serves as a cooperativity coordinator for RNAi.Item The Roles of Small and Long Non-Coding RNAs in Regulating Gene Expression(2014-01-23) Xue, Zhihong; McKnight, Steven L.; Liu, Yi; Liu, Qinghua; Conrad, NicholasRecent studies have revealed that a large proportion of a eukaryotic genome is transcribed into non-coding RNAs (ncRNAs). Based on size, these RNAs can be classified as small non-coding RNAs (sRNAs) and large non-coding RNAs (lncRNAs). The ncRNA regulatory networks control various levels of gene expression and play significant roles in diverse biological processes. Argonaute proteins, the core proteins in RNAi pathways, are required for the biogenesis of some sRNAs, including the PIWI-interacting RNAs and some microRNAs. How Argonautes mediate maturation of sRNAs independent of their slicer activity was not clear.The maturation of the Neurospora miRNA-like sRNA, milR-1, requires the Argonaute protein QDE-2, Dicer, and exonuclease QIP. Here, I reconstituted this Argonaute-dependent sRNA biogenesis pathway in vitro, and demonstrated that QDE-2 mediates milR-1 maturation by recruiting exosome and QIP, and by determining the size of milR-1. QIP first separates the QDE-2-bound duplex milR-1 precursor and then mediates 3’ to 5’ trimming and maturation of milR-1 precursor together with exosome using a hand-over mechanism. Our results establish a biochemical mechanism of an Argonaute-dependent sRNA biogenesis pathway and critical roles of exosome in sRNA processing. Natural antisense RNAs, which are mostly lncRNAs, are widely found in eukaryotic organisms and have been implicated in diverse physiological processes. The physiological importance of antisense RNAs and how they regulate sense RNAs are not clear. frequency (frq) encodes a core component of the Neurospora circadian oscillator. Here, I demonstrated that the simultaneous transcription of qrf, the long non-coding frq antisense RNA, represses frq transcription by inducing RNA polymerase II collision-triggered premature transcription termination and chromatin modifications. The expression of frq also inhibits the expression of qrf and surprisingly, drives the antiphasic rhythm of the qrf transcripts in the dark. The mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for robust and sustained circadian rhythmicity. Our results establish antisense transcription as an essential feature in a eukaryotic circadian system and demonstrate the importance and mechanism of antisense RNA action. Together, the studies described in this dissertation shed light on the mechanisms of gene expression regulated by sRNAs and lncRNAs.