RESUMO
Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.
Assuntos
Anticorpos , Recursos Comunitários , Humanos , Reprodutibilidade dos Testes , Diagnóstico por ImagemRESUMO
Kaposi's sarcoma (KS) is strongly associated with KS herpes virus infection, and inflammation plays an important role in this disease. We have shown that human KS biopsy-derived SLK cells, which are of endothelial origin and form KS-like tumors in nude mice, express the viral RNA pattern recognition receptors Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma-differentiation-associated gene 5 (MDA5). Furthermore, SLK cells have enhanced release of IL-6, IL-8 (CXCL8), RANTES (CCL5), and IP-10 (CXCL10) proteins in response to the synthetic viral RNA analog poly(I:C). SiRNA knockdowns demonstrated that TLR3 mediates this inflammatory response to poly(I:C) in SLK cells. Furthermore, knockdown of the RNA receptor RIG-I resulted in enhanced chemokine release, in a TLR3 pathway-dependent manner. Thus, exposure of KS cells to viral RNA ligands can result in a TLR3-mediated increase in the secretion of inflammatory proteins associated with KS cell growth that may contribute to disease.
Assuntos
RNA Helicases DEAD-box/metabolismo , Inflamação/metabolismo , RNA de Cadeia Dupla/imunologia , Sarcoma de Kaposi/metabolismo , Receptor 3 Toll-Like/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proteína DEAD-box 58 , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Inflamação/imunologia , Poli I-C/imunologia , RNA Mensageiro/análise , RNA Interferente Pequeno , Receptores Imunológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Kaposi/imunologia , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (approximately 0.5 kb of the approximately 1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.
Assuntos
Proteínas Fúngicas/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Telomerase/genética , Telomerase/metabolismo , Alelos , Sítios de Ligação , Proteínas de Ligação a DNA , Dimerização , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade , Telomerase/química , Moldes GenéticosRESUMO
It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown. By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S. cerevisiae RNA, we found a conserved region that is essential for telomere maintenance. Phylogenetic analysis and computer folding revealed that this region is conserved not only in primary nucleotide sequence but also in secondary structure. A common bulged-stem structure was predicted in all seven yeast species. Mutational analysis showed the structure to be essential for telomerase function. Suppression of bulged-stem mutant phenotypes by overexpression of Est1p and loss of co-immunoprecipitation of the mutant RNAs with Est1p indicated that this bulged stem is necessary for association of Est1p, a telomerase regulatory subunit. Est1p in yeast extract bound specifically to a small RNA containing the bulged stem, suggesting a direct interaction. We propose that this RNA structure links the enzymatic core of telomerase with Est1p, thereby allowing Est1p to recruit or activate telomerase at the telomere.