THE RNA COMPONENT OF TELOMERASE: AN UNUSUAL NONCODING RNA

Elongation of human telomeres requires the enzymatic activity of the telomerase holoenzyme. In vitro, telomerase activity can be reconstituted by the catalytic subunit (hTERT) and the RNA subunit (called hTERC, hTER, or hTR). However, the routes of the RNA and protein components of telomerase from their generation to assembly into telomerase appear to be complex and likely to be highly regulated. The expression of hTERT is greatly diminished in most human adult somatic cells, with some exceptions, notably germ cells and stem cells. In contrast, the expression of hTER is readily detectable in a wide range of normal cells. The biological significance of this accumulation of telomerase RNA in human cells that lack significant levels of telomerase enzymatic activity is not well understood. Also still largely unknown are how the telomerase components are regulated, how the RNP telomerase enzyme complex is assembled, and how such assembly is controlled. We investigated the cis-acting elements of telomerase RNA gene constructs to explore how they affect the journey of telomerase RNA from gene to telomerase activity in human cells. We analyzed expression and processing of human telomerase RNA in cultured transformed human cells, focusing on their dependence on cis-acting sequences in a variety of hTER gene constructs. Although this work was done in the course of experimenting with expression constructs designed with the goal of expressing human telomerase RNA efficiently in human tumor and other cells, the single-copy telomerase RNA gene is also an interesting small nuclear RNA (snRNA)-encoding gene for a number of reasons. First, in vertebrates, many small RNAs are encoded as multiple genes falling into gene families; single-copy snRNA genes are less common. Second, the transcriptional and processing machinery responsible for expression of telomerase RNA differs among different eukaryotic phyla. The transcription and expression of telomerase RNA have been characterized in diverse species, including ciliates, budding yeasts, and humans (Feng et al. 1995). It has been shown that ciliate telomerase RNA is transcribed by RNA polymerase III (pol III) (Yu et al. 1990; Romero and Blackburn 1991) and that the ciliate telomerase RNA promoter functionally resembles that of the vertebrate U6/7SK promoters, and also lacks cis-acting control elements that lie within the RNA-coding sequence. In this way, the ciliate telomerase RNA promoter is unlike the promoters of the 5S RNA and tRNA genes transcribed by RNA pol III (Romero and Blackburn 1991; Hargrove et al. 1999). In contrast to ciliates, in budding yeast and vertebrates, telomerase RNA expression is driven by an RNA pol II promoter. Third, the processing of telomerase RNA transcripts to yield the mature telomerase RNA competent for assembly into telomerase enzyme is not well understood. Ciliate telomerase RNA has a binding site for a telomerase-specific La family protein that is involved in its biogenesis and stability (Witkin and Collins 2004). In contrast, the mature processed form of budding yeast telomerase RNA contains a binding site for the Sm protein complex (Seto et al. 1999), which is involved in the biogenesis of spliceosomal snRNAs. In budding yeast, the RNA pol-II-transcribed telomerase RNA gene products include a polyadenylated transcript. Although normally present in very low amounts, in various mutants, this polyadenylated form of telomerase RNA accumulates relative to the mature RNA (Chapon et al. 1997). In contrast to the yeast telomerase RNA, studies of human telomerase RNA suggest that its transcript is nonpolyadenylated (Feng et al. 1995). Furthermore, in contrast to both ciliate and budding yeast telomerase RNAs, hTER contains a 3′ nucleolar RNA-like box H/ACA domain (Mitchell et al. 1999). This box H/ACA domain is conserved among vertebrate telomerase RNAs, as is the presence of a small Cajal-body-specific RNA (scaRNA) sequence (Richard et al. 2003). The detection of hTER in nucleoli (Wong et al. 2002) has further suggested that regulation of hTER biosynthesis and its incorporation into the telomerase RNP may be small nucleolar RNA (snoRNA)-like. Vertebrate Expression and Suppression of Human Telomerase RNA