A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase

Abstract

Telomerase maintains the simple sequence repeats at chromosome ends, protecting cells from genomic rearrangement, proliferative senescence and death. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble into active enzyme in a heterologous cell extract, but the physiological process of telomerase biogenesis is more complex. The endogenous accumulation of Tetrahymena thermophila TERT and TER requires an additional telomerase holoenzyme protein, p65. Here, we reconstitute this cellular pathway for telomerase ribonucleoprotein biogenesis in vitro. We demonstrate that tandem RNA interaction domains in p65 recognize the sequence of the TER 3′ stem. Notably, the p65-TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, we show that p65 enhances TERT-TER interaction by a mechanism involving a conserved bulge in the protein-bridging TER molecule. These findings reveal a pathway for telomerase holoenzyme biogenesis that preassembles TER for TERT recruitment.Cellular ribonucleoprotein (RNP) biogenesis has gained attention as a dynamic, regulated cascade of events that can be affected in human disease 1-3. RNPs assemble in vivo aided both by assembly chaperones, which contribute transient interactions, and by architectural RNA-binding proteins, which remain integral components of an assembled RNP 4 . Progress in the elucidation of physiological RNP biogenesis mechanisms has been impeded by the limited success in reconstitution of ordered protein and RNA assembly pathways in vitro.TERT and TER can assemble in rabbit reticulocyte lysate (RRL) to form a minimal recombinant telomerase RNP 5 . The physiological process of telomerase enzyme biogenesis, however, seems more complex. In vivo, human TER accumulation and telomere length maintenance are compromised by disease-associated single-residue substitutions in the RNA-binding protein dyskerin 6,7 . In budding yeast, TER accumulation requires RNAbinding proteins from the Sm family 8 . The potentially pleiotropic impact of vertebrate dyskerin or yeast Sm protein alterations complicates the interpretation of their direct roles in telomerase RNP assembly. Additionally, studies of these RNP assembly pathways in vitro are hampered by the inability to reconstitute a dyskerin or Sm RNP efficiently in the absence of other, typically concerted cellular events. In the biological context, it was thus unresolved whether TERT assembles with TER unassisted or only transiently chaperoned, or whether [9][10][11][12] . Genetic depletion of T. thermophila p65 reduces TER accumulation without impact on other small nuclear RNAs, suggesting that in contrast to vertebrate dyskerin or yeast Sm proteins, p65 has a telomerase-specific function in RNP biogenesis 9 . Based on these and additional findings, we proposed a p65-dependent pathway for the physiological biogenesis of T. thermophila telomerase RNP. Here, we test the proposed telomerase RNP assembly pathway by reconstituting the inter...

xRRM

“…Recently, we determined the solution and crystal structures of the C-terminal domain of Tetrahymena p65-TER-TERT, which contribute to different aspects of telomerase RNP assembly and function. 10,11,15,16 Some mutations in TER and TERT that abrogate or decrease activity in telomerase assembled in vitro from TERT and TER are rescued when p65 is present, which further underscores the essential role of p65 in the biogenesis and assembly of the telomerase holoenzyme. 15,17 The interaction of p65 with TER is discussed next in detail.…”

“…Genetic depletion of p65 in Tetrahymena leads to decreased levels of Although in vitro only TER and TERT are required to reconstitute telomerase catalytic activity, in vivo the RNP catalytic core contains p65 in addition to TER and TERT. 10,11 The p65 interacts with stem 4 (S4), UUU-3'OH and possibly stem 1 of TER, which are distinct from the TERT binding sites (Fig. 1B).…”

“…1B). [10][11][12][13][14] The interaction of p65 with TER is essential for catalytic core assembly in vivo 10,11 and enhances assembly and activity in vitro. 10,12,15 An extensive network of biochemical interactions has been established between the three RNP catalytic core components, Tetrahymena specific N-terminal domain (in cyan) that is absent in other LARP7 proteins.…”

“…5,8,9 both TER and TERT, indicating its critical role in Tetrahymena telomerase biogenesis and assembly. 8,10,11 p65 consists of four domains: N-terminal domain, La motif, RRM1 and C-terminal domain (Fig. 1A).…”

Structural study of elements of Tetrahymena telomerase RNA stem–loop IV domain important for function

“…The TERT protein contains a region of homology with other reverse transcriptases as well as telomerase-specific regions that are also required for activity, including the essential TEN (TERT essential N-terminal) and RBD (RNA binding domain) domains implicated in TER binding (Lingner et al 1997;Nakamura and Cech 1998;Lai et al 2001;O'Connor et al 2005;Jacobs et al 2006). Of the accessory proteins, p65 has been shown to form a complex with TER and enhance the assembly of TERT with TER in vitro (Prathapam et al 2005;O'Connor and Collins 2006). This role for p65 in RNP assembly is likely to account for its genetic requirement for TER and TERT accumulation in vivo.…”