Browsing by Subject "small interfering RNA"

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Distinct Antiviral Responses in Pluripotent versus Differentiated Cells
(PLOS Pathogens, 2014-02-06) Pare, Justin M.; Sullivan, Christopher S.
There is a mystery unfolding, the solution of which has implications for the understanding of both stem cell biology and the evolution of the vertebrate pathogen defense response. At the heart of this puzzle lies the observation of substantially different antiviral responses in mammalian cells with high potency (e.g., embryonic stem, oocytes, induced pluripotent, teratocarcinoma, and embryonic carcinoma cells) versus differentiated somatic cells (i.e., epithelial, fibroblast, lymphocyte). While differentiated cells are proficient in the interferon (IFN)-associated protein-based response [1]–[3], pluripotent cells have an attenuated IFN response [4]–[8]. Conversely, pluripotent cells can utilize RNA interference (RNAi) to combat viruses [9], [10], while this response is attenuated in differentiated cells [11]. Here we provide an overview of this developing area of virology.
A Human Torque Teno Virus Encodes a MicroRNA That Inhibits Interferon Signaling
(PLOS Pathogens, 2013-12-19) Kincaid, Rodney P.; Burke, James M.; Cox, Jennifer C.; de Villiers, Ethel-Michele; Sullivan, Christopher S.
Torque teno viruses (TTVs) are a group of viruses with small, circular DNA genomes. Members of this family are thought to ubiquitously infect humans, although causal disease associations are currently lacking. At present, there is no understanding of how infection with this diverse group of viruses is so prevalent. Using a combined computational and synthetic approach, we predict and identify miRNA-coding regions in diverse human TTVs and provide evidence for TTV miRNA production in vivo. The TTV miRNAs are transcribed by RNA polymerase II, processed by Drosha and Dicer, and are active in RISC. A TTV mutant defective for miRNA production replicates as well as wild type virus genome; demonstrating that the TTV miRNA is dispensable for genome replication in a cell culture model. We demonstrate that a recombinant TTV genome is capable of expressing an exogenous miRNA, indicating the potential utility of TTV as a small RNA vector. Gene expression profiling of host cells identifies N-myc (and STAT) interactor (NMI) as a target of a TTV miRNA. NMI transcripts are directly regulated through a binding site in the 3′UTR. SiRNA knockdown of NMI contributes to a decreased response to interferon signaling. Consistent with this, we show that a TTV miRNA mediates a decreased response to IFN and increased cellular proliferation in the presence of IFN. Thus, we add Annelloviridae to the growing list of virus families that encode miRNAs, and suggest that miRNA-mediated immune evasion can contribute to the pervasiveness associated with some of these viruses.
Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species
(PLOS One, 2014-03-05) Liu, Bigang; Badeaux, Mark D.; Choy, Grace; Chandra, Dhyan; Shen, Irvin; Jeter, Collene R.; Rycaj, Kiera; Lee, Chia-Fang; Person, Maria D.; Liu, Can; Chen, Yurping; Shen, Jianjun; Jung, Sung Yun; Qin, Jun; Tang, Dean G.
Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ~35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29–80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.