Identification of Two RNA-binding Proteins Associated with Human Telomerase RNA

Abstract

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo. INTRODUCTIONTelomerase is a specialized reverse transcriptase that is essential for telomere maintenance. The telomerase ribonucleoprotein (RNP) uses an internal RNA template to synthesize telomeric repeat sequences onto chromosome ends. Deletion of the essential RNA component of telomerase leads to progressive telomere shortening, chromosome instability, and cell death in both yeast and mouse cells (Singer and Gottschling, 1994;McEachern and Blackburn, 1996;Blasco et al., 1997;Lee et al., 1998).The telomerase enzyme is made up of an essential core as well as several accessory proteins. The core telomerase enzyme consists of the RNA component (telomerase RNA [TR]) and the catalytic subunit (telomerase reverse transcriptase [TERT]). The structure of the RNA component is conserved (Romero and Blackburn, 1991;Lingner et al., 1994) in ciliates where the RNA is 150 -200 nucleotides in length (Greider and Blackburn, 1989). In mammalian cells the RNA component is significantly larger, 390 -450 nucleotides (Blasco et al., 1995;Feng et al., 1995), and the structure is highly conserved among vertebrates (Chen, Blasco, and Greider, unpublished results). The catalytic TERT component, first identified in the ciliate Euplotes (Lingner and Cech, 1996), has homologues in yeast (EST 2), human (hTERT), and mouse (mTERT) (Harrington et al., 1997b;Lingner et al., 1997b;Meyerson et al., 1997;Nakamura et al., 1997;Greenberg et al., 1998;Martin-Rivera et al., 1998). TERT contains sequence motifs similar to the conserved core of reverse transcriptases, and mutation of the conserved essential aspartate residues in the catalytic triad of reverse transcriptases eliminates telomerase activity (Lingner et al., 1997b;Weinrich et al., 1997). Minimal telomerase activity can be reconstituted in an in vitro transcription/translation extract using just the TERT and TR components Be...

Telomerase dysfunction and dyskeratosis congenita

“…In a recent study by our group, no coding mutations in three candidate genes (GAR1 NHP2 and NOP10) were detected in a selection of DC families showing autosomal recessive inheritance (Marrone et al unpublished data). A similar study was undertaken with hStau, a doublestranded RNA binding protein associated with the telomerase complex in vivo (Le et al 2000). Again, our results revealed no mutations in the coding region of hStau that were associated with the disease (Walne et al unpublished data).…”

Isolation and characterization of Staufen-containing ribonucleoprotein particles from rat brain

“…Fig. 1A shows that both Staufen proteins are preferentially found in the P100 pellet because they associate with intracellular membranes (19) and ribosomes (14,(23)(24)(25)(26). In addition, a significant fraction of Stau1 (19,23,24) (labeled Stau1 63 ) and all three known differentially spliced isoforms of Stau2 (17) (labeled Stau2 62 , Stau2 59 , and Stau2 52 ) are present in the S100 (Fig.…”

In vivo identification of ribonucleoprotein-RNA interactions

“…4Cc is a positive control showing that phosphocAMP response element-binding protein is observed in the cells after BDNF treatment. Further, hnRNP A1, hnRNP C1͞C2, and ribosomal protein L22 were found to associate with telomerase mRNA (44)(45)(46) and have also been found to increase under BDNF-stimulated conditions. Because telomerase dysfunction has been associated with the onset of rheumatoid arthritis (47), and also because the same RBPs bind to ank RNA, both of which are associated with arthritic physiology, it is tempting to speculate that these RBPs may be involved in coordinating aspects of normal bone physiology.…”