Browsing by Subject "Immunoprecipitation"
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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 Characterization of the In Vivo Functions of Y-Family DNA Polymerases Kappa and Rev1(2008-09-18) Kosarek, Jayme Nicole; Friedberg, Errol C.DNA translesion synthesis (TLS) is a mode of damage tolerance utilized by cells experiencing replicative stress as a result of DNA damage. TLS is characterized by the synthesis of DNA opposite template lesions, a process that requires the function of specialized DNA polymerases. My studies focus on particular aspects of Rev1 and Polymerase kappa (Polκ) function in vivo. One of the main goals of my work was to characterize the conservation of the interaction between the Rev1 C-terminus and other Y-family polymerases (demonstrated in vertebrates) in lower eukaryotic species. I showed that these interactions are not conserved in the yeasts S. cerevisiae or S. pombe, nor in the nematode C. elegans, yet they are conserved in the fruit-fly Drosophila melanogaster. Furthermore, I experimentally determined the requirements of the Y-family polymerase interactions in Drosophila and mouse for comparative analysis. The results of this study concluded that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another. Another central goal of this work was to identify new Rev1 protein interaction partners in S. cerevisiae. I created a yeast two-hybrid library for screening with Rev1 bait. After identifying and verifying interaction partners for Rev1, I further investigated the epistatic relationship of these genes to Rev1 with respect to UV-radiation. The candidate genes investigated do not appear to function in a synonymous pathway to Rev1 in the response to UV-induced stress. A final goal of this work was to determine the spontaneous mutation frequencies of Polκ-/- mice using a well-validated in vivo mutation detection system. I found that somatic spontaneous mutation frequencies are elevated in 9 and 12-month old Polκ-/- kidney and liver, but not wild-type or Polη-/- tissues. Furthermore, I characterized the mutation spectra of these mice and observed a specific elevation in transversion mutations G:C>C:G and G:C>T:A. These data are consistent with previous observations of Polκ-/- mice, and hint at what types of spontaneous damage may be naturally occurring substrates for Polκ in vivo.Item Molecular and Functional Analysis of Phosphatidylinositol 4 Kinase Type II Beta(2008-09-12) Jung, Gwanghyun; Albanesi, Joseph P.Phosphoinositides play fundamental roles in controlling membrane-based signaling events. Phosphatidylinositol 4-kinases (PI4Ks) catalyze the production of PI4P, a major precursor in phosphoinositide biosynthesis, and consist of two classes (type II and type III), each divided into two isoforms (alpha and beta ). PI4KIIalpha and beta differ primarily in their distributions between cytosol and membranes: PI4KIIalpha is almost exclusively membrane-bound, by virtue of its palmitoylation in a cysteine-rich motif; although PI4KIIbeta also contains a palmitoylatable cysteine-rich motif, this isoform is almost evenly distributed between membranes and cytosol, and only about half of the membrane-associated pool is palmitoylated. My study focused on determining the functions of post-translational modifications and domains of PI4KIIbeta and on identifying its binding partners, with the long-term goal of understanding the roles and mechanisms of regulation of this kinase. Domain analysis shows that the C-terminal 160 amino acids of PI4KIIs determine the distinct membrane binding properties and activities of PI4KIIalpha and beta . As expected, based on our previous work with PI4KIIalpha , palmitoylation of PI4KIIbeta is important for its membrane binding and activity. Although PI4KIIbeta is also phosphorylated in cells, this modification has no detectable effect on any examined property of the kinase. Immunoprecipitation and mass spectrometry revealed a genuine binding partner for PI4KIIbeta , Hsp90. The functional significance of the Hsp90-PI4KIIbeta interaction was defined using geldanamycin, a specific Hsp90 inhibitor. Geldanamycin treatment disrupts the interaction and destabilizes PI4KIIbeta , reducing its half-life by 40% and increasing its susceptibility to proteasomal degradation. Although full-length PI4KIIalpha does not bind Hsp90, and is not destabilized by geldanamycin treatment, a cytosolic PI4KIIalpha truncation mutant becomes sensitive to geldanamycin and binds to Hsp90. Thus, both PI4KII isoforms contain Hsp90 binding sites but only PI4KIIbeta requires Hsp90 for stabilization, presumably because there is a substantial cytosolic pool of this isoform. Interestingly, brief exposure to geldanamycin causes a partial redistribution of PI4KIIbeta from the cytosol to membranes, which results in increased PI4P synthesis in cells. Moreover, the growth factors EGF and PDGF also disrupt the interaction between Hsp90 and PI4KIIbeta , suggesting that Hsp90 not only protects PI4KIIbeta from degradation, but may also prolong its residency in the cytoplasm until extracellular signals release Hsp90 from the kinase. Currently the precise roles of PI4KIIbeta are unknown. Based on its partial redistribution to the plasma membrane upon cell treatment with growth factors, I speculated that PI4KIIbeta may somehow be involved in receptor-mediated endocytic trafficking. My results, employing siRNA-based knockdown strategies, indicate that depletion of PI4KIIbeta enhances early steps of EGFR internalization and subsequent initiation of ERK activation in response to EGF treatment. The facilitated endocytosis that results from this depletion is likely due to an increase in endosomal fusion. Indeed, activities of the endosomal fusion facilitators EEA1 and Rab5 increase in PI4KIIbeta depleted cells. These results suggest for the first time a role of PI4KIIbeta in endocytic trafficking and signaling of the EGFR.Item Small RNAs Regulate Transcription by Interacting with Noncoding RNA Transcripts(2010-05-14) Schwartz, Jacob C.; Corey, David R.General methods for controlling gene expression have long been appreciated as an attractive target in drug design. Recently, the Corey lab has demonstrated that short RNA duplexes designed to target the promoter region for human genes can inhibit or activate gene expression in a sequence dependent manner. The mechanism by which RNAs achieve promoter recognition has remained unclear. Sequence specific recognition could be achieved by (1) RNA hybridization to genomic DNA, or (2) RNA recognition of some uncharacterized RNA species. Promoter targeted duplex RNA has been shown to recruit argonaute proteins to the promoter DNA and these proteins are necessary for duplex RNAs to regulate transcription. Argonaute proteins are known to recognize RNA:RNA interactions. However, genes targeted with duplex RNAs have no characterized transcripts in their promoters. I tested the hypothesis that promoter RNA transcripts exist and serve as a substrate for short duplex RNAs to hybridize to and regulate gene expression of adjacent genes. I found previously undiscovered RNA transcripts expressed from the promoter of progesterone receptor (PR) using RT-PCR. Quantitative RT-PCR of the promoter RNA of PR reveals expression levels between 10 and 1000 fold lower than PR in T47D and MCF7 breast cancer cells. I have cloned three transcripts overlapping the promoter of PR from two cell lines – T47D and MCF7, each with unique splicing and transcription start sites. All of these transcripts initiate within the protein coding region of PR and run antisense to the gene PR. I have been able to show that the promoter transcripts can be immunoprecipitated with antibodies against the argonaute proteins in cells transfected with duplex RNAs targeting the promoter of PR but not in cells transfected with mismatched duplex RNAs. Also, biotinylated RNAs bind to and pull down these noncoding RNAs. Finally, knockdown of the antisense transcript with an antisense oligonucleotide prevent gene activation by duplex RNAs. Following this study, our lab uncovered that duplex RNAs can target beyond the 3' terminus of genes and silence or activate transcription. I further showed that this transcription regulation is mediated by argonaute binding to noncoding RNAs overlapping the 3' terminus of the genes, PR and BRCA1. The signal is transmitted from the 3' terminus to the gene promoter because the 5' and 3' ends of these genes are held in a chromatin loop, which I validated using a chromatin conformation capture assay. This brings the ends of the gene in close proximity to each other. Due to this interaction, short RNAs that bind a noncoding RNA at the 3' end of the gene also physically interacts with noncoding RNAs that associate with the gene promoter. This is confirmed by RNA immunoprecipitation of both transcripts with duplex RNAs targeting either the 5' or 3' ends of the gene. More than 20 years ago, it was found that proteins recognizing DNA at the 5’ end of genes could regulate transcription. This study presents a paradigm shift implicating noncoding RNAs at the 5' and 3' ends of genes can be recognized by proteins which activate or inhibit transcription of adjacent protein coding genes. Recent studies demonstrate an abundance of RNAs transcribed in human cells that do not code for protein. My results suggest a new model for duplex RNA recognition of gene promoters. Argonaute proteins loaded with one strand of the RNA duplex recognizes, through Watson-Crick base pairing, a noncoding transcript that is associated with chromatin at the promoter of the targeted gene. This RNA:RNA interaction in close proximity to the promoter mediates protein-protein interactions between argonaute and other factors on the promoter to turn off or on gene expression.