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.

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 (ϳ0.5 kb of the ϳ1.3-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.Telomerase is a ribonucleoprotein enzyme that catalyzes the addition of telomeric DNA sequence to chromosome ends. Telomerase minimally contains a catalytic subunit, which is a reverse transcriptase (19), and an RNA subunit that provides the template for telomeric DNA addition (9). In the budding yeast Saccharomyces cerevisiae, the telomerase RNA subunit is approximately 1.3 kb and is encoded by the TLC1 gene (38). The function of less than 80 nucleotides (nt) of TLC1 RNA has been reported. The goals of the present study are to determine the regions of TLC1 RNA that are required for in vivo telomerase function and to then use this information to facilitate the study of RNA-protein interactions in this complex.The S. cerevisiae telomerase catalytic subunit, encoded by the EST2 gene, and TLC1 RNA comprise the catalytic core of the enzyme and are required for activity in vitro (6, 19). Additional proteins encoded by EST1, EST3, and CDC13 are required in vivo for telomere maintenance. Mutations in EST2, TLC1, EST1, EST3, and CDC13 can lead to an est (ever shorter telomeres) phenotype-telomeres progressively shorten, and the cells eventually senesce (14, 21). Est1p and Est3p are telomerase subunits (11), while Cdc13p binds single-stranded telomeric DNA and appears to be a component of the telomere itself (12,16,25). Est1p also binds single-stranded G-rich telomeric DNA in vitro (40) and interacts with Cdc13p by two-hybrid analysis (29). The Est1p-Cdc13p interaction recruits telomerase to the telomere (7, 26).TLC1 RNA has been proposed to bind Est1p via an RNA recognition motif (44). TLC1 RNA contains a 48-nt stem-loop that interacts either directly or indirectly with the DNA endbinding protein Ku (27). In addition, TLC1 RNA contains an Sm binding site near its 3Ј end that is important for the stability of the RNA and is bound by Sm proteins, therefore leading to the classification of S. cerevisiae telomerase as an Sm snRNP (small nuclear ribonucleoprotein particle) (35). Because TLC1 RNA intera...

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.

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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.