Biological and biochemical functions of RNA in the tetrahymena telomerase holoenzyme

Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.     

Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei

Messenger RNA maturation in trypanosomes involves an RNA trans-splicing reaction in which a 39 nucleotide 5'-spliced leader (SL), derived from an independently transcribed 139 nucleotide SL RNA, is joined to pre-mRNAs. Trans-splicing intermediates are structurally consistent with a mechanism of SL addition which is similar to that of cis-splicing of nuclear pre-mRNAs; homologous components (e.g. the U small nuclear RNAs) exist in both cis- and trans-splicing systems, suggesting that these also participate in the two types of splicing reactions. In this study, ribonucleoprotein (RNP) complexes containing the trypanosome SL and U2 RNAs were purified and characterized. Although present at low levels in cellular extracts, the SL and U2 RNPs are the two most abundant of the several non-ribosomal small RNP complexes in these cells. The purification scheme utilizes ion-exchange chromatography, equilibrium density centrifugation, and gel filtration chromatography and reveals that the SL RNP shares biophysical properties with U RNPs of trypanosomes and other eukaryotes; its sedimentation coefficient in sucrose gradients is approximately 10 S, and it is resistant to dissociation during Cs2SO4 equilibrium density centrifugation. Complete separation of the SL and U2 RNPs was achieved by non-denaturing polyacrylamide gel electrophoresis. Proteins purifying with the SL and U2 RNPs were identified by 125I-labeling of tyrosine residues. Four SL RNP proteins with approximate molecular masses of 36, 32, 30, and 27 kDa and one U2 RNP protein of 31 kDa were identified, suggesting that different polypeptides are associated with these two RNAs. These particles are not immunoprecipitated by anti-Sm sera which recognizes U snRNP proteins of other eukaryotes including humans plants and yeast.     

Biochemical properties of Trypanosoma cruzi telomerase

Trypanosomatid parasite infections have a devastating impact on human health. Little is known about the requirements for parasite growth during any stage of their complex, multi-host life cycle. In most eukaryotic organisms, sustained cell proliferation requires telomerase-dependent telomere length maintenance. Here we investigate the regulation and biochemical features of telomerase from Trypanosoma cruzi, the causative agent of Chagas disease. We found that T.cruzi telomerase is active in extracts from multiple developmental stages of the parasite life cycle. Detailed characterization of the enzymatic properties of telomerase using epimatigote-stage extract revealed a unique combination of substrate specificities, consistent with the evolutionary divergence of trypanosomes from previously established model systems for telomerase biochemical characterization. Results from partial purification of T.cruzi telomerase suggest that the catalytically active enzyme is a large ribonucleoprotein complex and that the internal RNA template has an atypical, cytosine-rich permutation. These results expand our understanding of telomerase enzymology and should encourage the development of parasite-specific telomerase inhibitors as a method for disease therapy.     

Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro

We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.     

RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

Telomerase synthesizes telomeric DNA repeats onto chromosome termini from an intrinsic RNA template. The processive synthesis of DNA repeats relies on a unique, yet poorly understood, mechanism whereby the telomerase RNA template translocates and realigns with the DNA primer after synthesizing each repeat. Here, we provide evidence that binding of the realigned RNA/DNA hybrid by the active site is an essential step for template translocation. Employing a template-free human telomerase system, we demonstrate that the telomerase active site directly binds to RNA/DNA hybrid substrates for DNA polymerization. In telomerase processivity mutants, the template-translocation efficiency correlates with the affinity for the RNA/DNA hybrid substrate. Furthermore, the active site is unoccupied during template translocation as a 5 bp extrinsic RNA/DNA hybrid effectively reduces the processivity of the template-containing telomerase. This suggests that strand separation and template realignment occur outside the active site, preceding the binding of realigned hybrid to the active site. Our results provide new insights into the ancient RNA/DNA hybrid binding ability of telomerase and its role in template translocation.