Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase catalytic subunit (hTERT) contributes to cell physiology independent of its ability to elongate telomeres. Here we show that hTERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome Cartilage-Hair Hypoplasia. hTERT and RMRP form a distinct ribonucleoprotein complex that exhibits RNA dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer-dependent manner. These observations identify a mammalian RdRP composed of hTERT in complex with RMRP.

Recent work has identified a subset of cells resident in tumors that exhibit properties similar to those found in normal stem cells. Such cells are highly tumorigenic and may be involved in resistance to treatment. However, the genes that regulate the tumor initiating cell (TIC) state are unknown. Here, we show that overexpression of either of the nucleolar GTP-binding proteins nucleostemin (NS) or GNL3L drives the fraction of genetically defined tumor cells that exhibit markers and tumorigenic properties of TICs. Specifically, cells that constitutively express elevated levels of NS or GNL3L exhibit increased TWIST expression, phosphorylation of STAT3, expression of genes that induce pluripotent stem cells, and enhanced radioresistance; in addition, they form tumors even when small numbers of cells are implanted and exhibit an increased propensity to metastasize. GNL3L/NS forms a complex with the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] and the SWItch-Sucrose NonFermentable (SWI-SNF) complex protein brahma-related gene 1 (BRG1), and the expression of each of these components is necessary to facilitate the cancer stem cell state. Together, these observations define a complex composed of TERT, BRG1, and NS/GNL3L that maintains the function of TICs.

fIn the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin is maintained by an RNA-directed RNA polymerase complex (RDRC) and the RNA-induced transcriptional silencing (RITS) complex in a manner that depends on the generation of short interfering RNA. In association with the telomerase RNA component (TERC), the telomerase reverse transcriptase (TERT) forms telomerase and counteracts telomere attrition, and without TERC, TERT has been implicated in the regulation of heterochromatin at locations distinct from telomeres. Here, we describe a complex composed of human TERT (hTERT), Brahma-related gene 1 (BRG1), and nucleostemin (NS) that contributes to heterochromatin maintenance at centromeres and transposons. This complex produced doublestranded RNAs homologous to centromeric alpha-satellite (alphoid) repeat elements and transposons that were processed into small interfering RNAs targeted to these heterochromatic regions. These small interfering RNAs promoted heterochromatin assembly and mitotic progression in a manner dependent on the RNA interference machinery. These observations implicate the hTERT/BRG1/NS (TBN) complex in heterochromatin assembly at particular sites in the mammalian genome.T elomeres and centromeres are both tightly condensed heterochromatic areas within the genome, and the maintenance of heterochromatin is important for overall genome stability. In Schizosaccharomyces pombe, heterochromatin near centromeres is maintained by the RNA-directed RNA polymerase complex (RDRC) and the RNA-induced transcriptional silencing (RITS) complex (1, 2). Specifically, inhibition of RNA-dependent RNA polymerase (RdRP) activity leads to loss of small interfering RNAs (siRNAs) that are associated with the RITS complex and correlates with loss of transcriptional silencing and heterochromatin at centromeres (3). In addition, when RdRP activity is inhibited, siRNAs that are usually associated with the RITS complex are lost (4). These observations implicate RdRPs as a component of a loop coupling heterochromatin assembly to siRNA production.In Caenorhabditis elegans, the Argonaute CSR-1, the RdRP EGO-1, and the Dicer-related helicase DRH-3 localize to chromosomes and are required for proper chromosome segregation, and in the absence of these factors, chromosomes fail to properly align in mitotic phase (5, 6). Moreover, a conserved germ line-specific nucleotidyltransferase, CDE-1, localizes specifically to mitotic chromosomes in embryos in a manner that requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1 (5, 6). Although it is clear that RdRP and components of the RNA interference (RNAi) machinery are necessary to regulate heterochromatin in S. pombe and C. elegans, it is believed that heterochromatin is regulated in mammals through different mechanisms (7).Telomerase is a ribonucleoprotein complex that elongates telomeres. Human telomerase reverse transcriptase (hTERT) acts as an RNA-dependent DNA polymerase (RdDP) and synthesizes telomere DNA from a nonco...

RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such as Caenorhabditis elegans and Neurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAs de novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However, de novo synthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5=-triphosphorylated ends, which indicates de novo synthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity.T elomerase reverse transcriptase (TERT) is known as the catalytic subunit of telomerase and is expressed at high levels in cancer cells but only at low levels in normal human somatic cells. TERT elongates telomeres by its RNA-dependent DNA polymerase (RdDP) activity, using the telomerase RNA component (TERC) as the template. TERT and TERC assemble and form telomerase; however, there is a population of TERT proteins that are not assembled into the telomerase complex (1). Several lines of evidence indicate that TERT plays roles independent of telomere maintenance; therefore, nonassembled TERT may be involved in complexes other than telomerase.RNA silencing is a sequence-specific gene regulatory mechanism mediated by double-stranded RNAs (dsRNAs). RNAdependent RNA polymerase (RdRP) is a key player in RNA silencing, and the polymerase is found in a variety of organisms, including fungi, plants, and worms (2). Although insects and mammals lack sequence homologues of cell-encoded RdRPs, phylogenetic and structural analyses of TERT revealed that TERT is closely related to RdRPs of RNA viruses as well as to retroviral RdDPs (3). In fact, we found that TERT generates dsRNA in a primer-dependent manner and works as an RdRP by a mechanism similar to that for cell-encoded RdRPs (4, 5). Both viral RdRPs and cell-encoded RdRPs transcribe singlestranded RNA (ssRNA) from template RNA, not only in a primer-dependent manner but also in a primer-independent manner. However, primer-independent initiation of RNA synthesis by TERT, a human RdRP, remains to be elucidated.To analyze the characteristics of the RdRP activity of human TERT, we established an in vitro RdRP assay in which we analyzed the RdRP activity of TERT immune complexes immunoprecipitated from cell lysates by use of an anti-human TERT monoclonal antibody (MAb) (IP-RdRP assay) (5). Here we investigated the detailed characteristics of RNAs processed through the IP-RdRP assay. The results indicate that TERT RdRP produces short RNAs in a primer-independent manner. The relationship between TERT protein levels and the RdRP acti...

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.