Cell Cycle-dependent Recruitment of Telomerase RNA and Cajal Bodies to Human Telomeres

Abstract

Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10 -40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance. INTRODUCTIONTelomeres are specific nucleoprotein complexes protecting the termini of eukaryotic linear chromosomes from degradation, end-to-end fusion and undesired recombination (for recent reviews, see Wong and Collins, 2003;Ferreira et al., 2004;Smogorzewska and de Lange, 2004;Blasco, 2005). In humans, the DNA component of telomere is made up of ϳ5-15 kb of double-stranded 5Ј-TTAGGG-3Ј repeats that terminates in a 3Ј overhang of ϳ50 -300 bases (Makarov et al., 1997;Wright et al., 1997). By invading into proximal double-stranded telomeric sequences, the single-stranded overhang supports formation of a large duplex loop, termed the T-loop (Griffith et al., 1999). Because of the unidirectionality of conventional DNA polymerases, the ends of telomeres cannot be fully duplicated and human telomeres lose ϳ50 -200 base pairs during each cell division cycle and eventually, critically short telomeres induce cell cycle arrest called proliferative senescence. Therefore, by imposing a limit on the replicative life span of somatic cells, telomere erosion represents an innate mechanism for tumor suppression (Smogorzewska and de Lange, 2004).The replicative telomere erosion can be balanced by the telomerase reverse transcriptase that adds telomeric DNA repeats to the 3Ј overhang of telomeres (reviewed in Collins and Mitchell, 2002;Cong et al., 2002). In humans, telomerase activity is not detectable in most somatic cells, but germ line and other highly proliferative cells as well as the majority of tumor-derived cell lines possess highly active telomerase, indicating that maintenance of telomere length is necessary for indefinite proliferation of human cells (Kim et al., 1994;Greider, 1996;Shay and Bacchetti, 1997). Telomerase is a ribonucleoprotein (RNP) enzyme that is minimally composed of the telomerase RNA (TR) that spec...

Cell Biology of the Arabidopsis Nuclear siRNA Pathway for RNA-directed Chromatin Modification

“…Cajal bodies can also be found in association with histone gene loci (Shopland et al 2001), consistent with the fact that Cajal bodies contain the U7 RNA that is required for 3′-end formation of histone mRNAs, which lack poly(A) tails (Liu et al 2006). Telomerase RNP maturation and trafficking also appear to involve Cajal bodies, as well as the nucleolus, en route to interactions with the telomeres that cap the ends of eukaryotic chromosomes (Jady et al 2006;Tomlinson et al 2006). Collectively, these studies implicate Cajal bodies in a wide array of RNA-mediated processes.…”

Coilin: The first 25 years

“…Early experiments detected telomerase RNA in CBs, and CBs associate with telomerase during Sphase. [38][39][40][41][42] Interestingly, telomerase RNA localization to CBs seems to be humanspecific because telomerase RNA was not found in mouse CBs. 43 Telomerase RNA is retained in CBs via protein WRAP53, which binds the CAB box sequence found in telomerase RNA and many scaRNAs.…”

Replication of Telomeres and the Regulation of Telomerase

“…CBs also harbor a class of CB-specific RNAs (scaRNAs) that are involved in methylation and pseudouridylation of small nuclear RNAs. In S phase of the cell cycle, a subset of CBs together with telomerase RNA is seen in association with telomeres in human cells (Jady et al 2006;Tomlinson et al 2006). As with other scaRNAs, human telomerase RNA contains a CAB-box motif that mediates association with CBs (Jady et al 2004).…”