We are also grateful to Jung Ro Lee for providing the -dyskerin antibody

We are also grateful to Jung Ro Lee for providing the -dyskerin antibody. chromosome end protection. Rather, it Adapalene physically associates with the telomerase RNP and serves as a positive regulator of telomerase activity in vivo (11). Although TERT can readily be identified by its conserved reverse transcriptase motifs, TER has diverged dramatically and exhibits little sequence similarity and vastly different sizes, ranging from 150?nt in (12) to ?1,200?nt in yeast (13C15). Phylogenetic and mutational analyses reveal functionally conserved elements within TER including Rabbit Polyclonal to Chk2 (phospho-Thr387) a single-stranded templating domain typically corresponding to one and a half telomeric repeats flanked by a 5 boundary element and a 3 pseudoknot domain Adapalene (16C19). Vertebrate TERs harbor a box H/ACA snoRNA motif, which binds dyskerin and is required for RNP maturation and nuclear localization (20). Telomerase activity can be reconstituted in vitro with TERT and TER (16, 21, 22), although additional proteins assemble with the core telomerase RNP in vivo. For example, Est1 is a noncatalytic component of the yeast telomerase that facilitates enzyme recruitment/activation at chromosome ends (23, 24). Relocation of the Est1 binding site to a different position in TER does not diminish telomerase activity in vivo (25), indicating that TER is a modular, highly flexible scaffold for telomerase proteins. The Ku70/80 heterodimer is reported to bind Adapalene TER in budding yeast and vertebrates (26, 27), but not in fission yeast (15). Ku promotes telomerase recruitment to nontelomeric DNA for de novo telomere formation in (28); its role in vertebrate telomere maintenance is unknown. We report the discovery of two distinct TER subunits in Adapalene (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”HQ401284″,”term_id”:”315630505″,”term_text”:”HQ401284″HQ401284) encodes a Adapalene putative template sequence (5-CTAAACCCTA-3) corresponding to 1 1.5 copies of the Arabidopsis telomere repeat (Fig.?1are indicated. Northern blot (cell culture are shown. The probes for Northern blotting and primer extension were directed at unique regions in the 5 and 3 ends of TER1, respectively. Closed triangles, TER1 transcript; open triangle, transcript that appears to be derived from AT1G71310, the protein encoding gene into which TER1 is embedded. In vitro transcribed TER1 served as a control for and (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”HQ401285″,”term_id”:”315630506″,”term_text”:”HQ401285″HQ401285), was also uncovered (Fig.?1and and encodes a 748-nt RNA and and and Fig.?S3). Unlike human (30) and yeast TER (31), we found no evidence of a poly-A tail for either Arabidopsis RNA. TER1 and TER2 contain a 220-nt stretch of 90% identity (Fig.?1and cell culture, positive control; buffer, negative control. (transcript containing a 10-nt sequence that could serve as putative template (415RNA); boiled reconstituted RNPs and total protein extract from cell culture. (and and and and and and and and and and and and Fig.?S4and Fig.?S4line (SAIL_556_A04), the T-DNA insertion lies directly within the templating domain of TER2 (Fig.?1(Fig.?3background. In this setting, TER2 was undetected and TER1 was reduced 3-fold in T1 plants and 24-fold in T2 (Fig.?3and Fig.?S4mutants and telomere length was similar in plants deficient in TER1 or both TER1/TER2, we conclude that TER1, not TER2, is required for telomere maintenance. To examine the templating function of TER1 in vivo, site-directed mutagenesis was used to mutate the 5-CUAAACCCUA-3 sequence in TER1 to 5-CUAAAGGCUA-3 (TER1CC). In vitro reconstitution reactions revealed that mutant RNA assembled into an active RNP and synthesized TTTAGCC repeats (Fig.?4and Fig.?S5and and ref. 38) was then employed to ask if TER1 specifies the telomere repeat sequence on chromosome ends. Using a subtelomeric primer directed at the left arm of chromosome 1, PETRA products were obtained from wild-type plants and transformants expressing 35S??TER1CC (Fig.?4and Fig.?S7A), indicating that TER1-POT1a binding is specific. Gel shift assays produced results consistent with the IP: TER1 formed an RNP complex with POT1a, but not POT1b (Fig.?5and Fig.?S6and ?and55and Fig.?S6and Fig.?S6and and Fig.?S6cell culture extracts using the indicated antibodies. Preimmune serum, and antihistone H3 antibodies were used as negative controls. (TER (12), and the minimal TERs defined for human (210?nt) (41) and (170?nt) (42). Thus, both TER1 and TER2 have the potential to function as a template for telomerase in vivo. Despite their similarities, the Arabidopsis TERs differ dramatically in overall nucleotide sequence.