2016 May 19
Active Yeast Telomerase Shares Subunits With Ribonucleoproteins RNase P and RNase MRP
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Active Yeast Telomerase Shares Subunits With Ribonucleoproteins RNase P and RNase MRP
Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs.
Copyright © 2016 Elsevier Inc. All rights reserved.
Figure 1. Mass spectrometry of endogenously expressed telomerase RNPs identifies the Pop1/Pop6/Pop7 proteins as part of active telomerase complexes
(A) Fractions from indicated steps during the purification as analyzed by western blotting. Blots were probed with anti Myc-antibodies (top) or anti-ProA antibodies (bottom). IN: input protein extract; FT: proteins in flow through; W1 BE: proteins retained on beads after the first wash; Fin BE: final proteins on the beads fraction that was used for mass spectrometry. The
TLC1 alleles used are indicated on top. (B) Left: Telomerase activity assays from cells expressing a ProA-Est2 protein and enriched with IgG as positive control. Right: Final telomerase activity on the IgG beads using the extracts from the cells as in panel A. +/− RNase: sample treatment with RNase A. 0+P 32: 32P end-labeled substrate oligo. Band labeled with a star is a background band migrating at the +3 position. (C) Combined results from three independent mass spectrometry determinations. Proteins identified are listed in order of the sum of peak-intensities (right column). The total number of unique peptides for each protein and the total protein coverage are indicated. Only proteins that were detected exclusively in the MS2-tagged Tlc1 fraction were considered and the top eight proteins are listed. For a more extensive list, see Figure S1A.
Figure 2. Binding of the Pop1/Pop6/Pop7 proteins to the Tlc1 RNA is specific
(A) Northern analysis of immunoprecipitations using IgG covered beads with extracts from strains expressing the indicated TAP-tagged proteins. For the HA
3-Pop1, anti-HA antibodies were used. The blots were hybridized with probes specific for the Tlc1 RNA and the Nme1 RNA at the same time. Ctrl RNAs on left: total RNAs from a wt strain or a strain that carried a tlc1Δ allele. IN: RNA from the input fraction IP: RNA from the immunoprecipitates; FT: RNA extracted from the unbound fraction. (B) Northern analysis as performed in panel (A). (C) Telomerase activity enrichment using a tagged Pop1, Pop6, or Pop7 protein. Top: antibodies used for immunoprecipitations with extracts derived from strains harboring the indicated tagged proteins. ProA-Est2 serves as positive control. Note that direct and indirect anti-HA refers to whether the anti-HA antibody was directly coupled to magnetic beads or whether antibody was first mixed with the slurry and then immunopurified with Protein A/G coupled magnetic beads. For the HA-tagged Pop1 protein, the latter technique appears more efficient in telomerase recuperation. Labeling as in Figure 1B. See also Figure S1B.
Figure 3. The CS2a/TeSS domain is a P3-like domain in Tlc1
(A) Schematic representation of the TeSS structure at the distal end of stem IVc of Tlc1 (left), the P3 domain of Nme1 RNase MRP RNA (middle) and the P3 domain of the Rpr1 RNase P RNA (right). Dark blue shading: identical nucleotides, light blue: purines and pyrimidines are conserved. Purple blue line on left of Tlc1: CS2a (Conserved Sequence element 2a; Gunisova et al., 2009). Dark green line on Nme1 RNA: nucleotides protected by Pop6/Pop7 binding (Perederina et al., 2007). For more details, see Figure S2A). (B) Telomere length analyses in strains harboring the indicated
TLC1 alleles. TLC1: wild type; Δ: tlc1Δ; ΔL: tlc1-ΔL allele that lacks the TeSS/P3 domain (see Figure S4); P3NME1: tlc1::P3NME1, the TeSS/P3 domain in the Tlc1 RNA was replaced with the one from the Nme1 RNA; P3RPR1; tlc1::P3RPR1, the TeSS/P3 domain in Tlc1 was replaced with the one from the Rpr1 RNA. +: Strain carried a wt TLC1 gene on a URA3 plasmid. Black wedges indicate outgrowth of strains after loss of the plasmid borne TLC1 gene; last lane reflects growth for 110 generations. Schematic structures on top are color coded as the nucleotides in panel (A). (C) Growth assays of cells that contain the indicated NME1 alleles: nme1Δ, complete deletion of NME1; ΔP3: nme1-ΔP3 allele that lacks the P3 domain; nme1::P3TLC1 the NME1 P3 domain was replaced with the TLC1 TeSS/P3 domain. Bottom plate: all strains contained a wt NME1 gene on a plasmid with the URA3 marker. Top plate: growth of cells after loss of the URA3, NME1 containing plasmid. Schematic structures and color coding as in panel A.
Figure 4. A cold sensitive allele of the
NME1 P3 domain confers cold-sensitive telomere shortening
Telomere length analyses in strains harboring the indicated
TLC1 alleles. Lanes 1–4: TLC1; lanes 5–8: tlc1Δ; lane 9–12: tlc1-ΔL; lanes 13–16: tlc1-ΔL; lanes 17–20: tlc1-P3NME1; lanes 21–24: tlc1-P3nme1-11. Schematics of stem IVc structures and color coding as in Figure 3 (see also Figure S4A). +: Strain carried a wt TLC1 gene on a URA3 plasmid. Black triangles: growth of cells after loss of the wt TLC1 gene at 30°C for 30 and 110 generations. Lanes with the blue star: strains were grown at 18°C for 110 generations. The red/blue bar at bottom indicates the temperatures the respective strain was grown at.
Figure 5. Binding characteristics of the Pop6/Pop7 heterodimer to the Tlc1 P3 domain
in vitro and in vivo
(A) Binding curves and apparent Kds of the Pop6/Pop7 heterodimer binding to indicated RNA oligonucleotides (see predicted structures, complete binding curve and gel assays in Figure S3). (B) Northern blot analysis of co-immunoprecipitated RNAs using IgG beads and extracts of strains that harbored TAP-tagged Pop7, a wt Tlc1 RNA and a 400 nt longer MS2-tagged Tlc1 RNA. The latter either contained a wt stem IVc, lanes 3–5; lacks the most distal stem-loop, MS2-
tlc1-ΔS: lanes 6–8; or lacks the TeSS/P3 completely, MS2- tlc1-ΔL, lanes 9–11. IN: input (2.5%); IP: immunoprecipitates (10%); FT: flowthrough (2.5%). (C) Western blot of input (left) and immunoprecipitates (right) from strains harboring a Myc 13-tagged Sme1 protein and Pop7-TAP. TCL1 alleles as indicated. See Figure S4A for predicted structures of the stem IVc in these mutated RNAs.
Figure 6. The Tlc1 TeSS/P3 domain is required for telomerase RNP integrity and function
in vivo and in vitro
(A) Western blots of input (left) and IPs (right) with extracts from strains harboring a HA
3-tagged Sme1 protein and in which the Est1 as well as Est2 proteins carried a Myc 12-tag. TLC1 alleles as indicated. Below, Northern blot of RNA extracted from equal amounts of the inputs (left) or IPs (right) as used for the Western, and which was hybridized to a TLC1-specific probe. (B) Telomerase activity assays with in vitro reconstituted RNPs using the Mini-T RNA (Zappulla et al., 2005). ProA-Est2 levels after the RRL reaction are indicated below. Recombinant rPop6/Pop7 heterodimer and/or rPop1 proteins were added as indicated. After the RRL assay, telomerase was enriched using IgG beads and activity was assayed as in Figure 1. Bar graph depicts quantified telomerase activities standardized to the one obtained without Pop protein addition (lane 2; * p < 0.05 as determined in an unpaired t-test with Welch’s correction). (C) Telomerase activity assays with in vitro reconstituted RNPs using a full-length wt Tlc1 RNA. Indications as in (B).
Figure 7. Model for core structure of the active yeast telomerase RNP
(A) Telomerase activity assays with
in vitro reconstituted RNPs using the Mini-T RNA, increasing amounts of Pop1 with or without Pop6/Pop7 addition as indicated. Final Pop1 concentrations are 0 (lanes 3, 7); 0.11 μM (lanes 4, 8); 0.33 μM lanes 5, 9) and 1 μM lanes 6, 10). Quantification of relative telomerase activities is indicated on the right. No additional protein addition (lane 3) was set as 1; data are averages from two experiments. (B) Binding of the Pop6/Pop7 and Pop1 proteins are modeled on top of the newly identified TLC1 P3 domain of the yeast telomerase RNA. The presence of the Tlc1 P3 domain provides a protein binding platform that keeps the essential Est1 and Est2 proteins on the active RNP. Note that physical interactions between the Pop-proteins and Est1 or Est2 as well as the placement of Est3 in the model are hypothetical.
All figures (7)
Specific Binding of a Pop6/Pop7 Heterodimer to the P3 Stem of the Yeast RNase MRP and RNase P RNAs
A Perederina et al.
RNA 13 (10), 1648-55.
Pop6 and Pop7 are protein subunits of Saccharomyces cerevisiae RNase MRP and RNase P. Here we show that bacterially expressed Pop6 and Pop7 form a soluble heterodimer tha …
The Yeast Telomerase Module for Telomere Recruitment Requires a Specific RNA Architecture
N Laterreur et al.
RNA 24 (8), 1067-1079.
Telomerases are ribonucleoprotein (RNP) reverse transcriptases. While telomerases maintain genome stability, their composition varies significantly between species. Yeast …
Footprinting Analysis of Interactions Between the Largest Eukaryotic RNase P/MRP Protein Pop1 and RNase P/MRP RNA Components
RD Fagerlund et al.
RNA 21 (9), 1591-605.
Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, a …
Of Proteins and RNA: The RNase P/MRP Family
O Esakova et al.
RNA 16 (9), 1725-47.
Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA co …
Structural and Functional Similarities Between MRP and RNase P
R Reddy et al.
Mol Biol Rep 22 (2-3), 81-5.
RNase P, the enzyme response for 5'-end processing of tRNAs and 4.5S RNA, has been extensively characterized from E. coli. The RNA component of E. coli RNase P, without t …
PubMed Central articles
Identification of Telomerase RNAs in Species of the Yarrowia Clade Provides Insights Into the Co-Evolution of Telomerase, Telomeric Repeats and Telomere-Binding Proteins
F Červenák et al.
Sci Rep 9 (1), 13365.
Telomeric repeats in fungi of the subphylum Saccharomycotina exhibit great inter- and intra-species variability in length and sequence. Such variations challenged telomer …
tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs
AK Hopper et al.
Front Genet 10, 96.
This article focuses upon gene products that are involved in tRNA biology, with particular emphasis upon post-transcriptional RNA processing and nuclear-cytoplasmic subce …
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Q Chen et al.
Int J Mol Sci 20 (2).
Cyclophilins (CYPs) are a member of the immunophilin superfamily (in addition to FKBPs and parvulins) and play a significant role in peptidyl-prolyl
cis- trans …
Roles of Telomere Biology in Cell Senescence, Replicative and Chronological Ageing
J Liu et al.
Cells 8 (1).
Telomeres with G-rich repetitive DNA and particular proteins as special heterochromatin structures at the termini of eukaryotic chromosomes are tightly maintained to safe …
TERribly Difficult: Searching for Telomerase RNAs in Saccharomycetes
M Waldl et al.
Genes (Basel) 9 (8).
The telomerase RNA in yeasts is large, usually >1000 nt, and contains functional elements that have been extensively studied experimentally in several disparate specie …
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Endoribonucleases / chemistry
Endoribonucleases / metabolism
Ribonuclease P / chemistry
Ribonuclease P / metabolism
Ribonucleoproteins / chemistry
Ribonucleoproteins / metabolism
Saccharomyces cerevisiae Proteins / chemistry
Saccharomyces cerevisiae Proteins / metabolism
Saccharomycetales / enzymology
POP1 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
EST1 protein, S cerevisiae
EST2 protein, S cerevisiae
mitochondrial RNA-processing endoribonuclease
POP6 protein, S cerevisiae
POP7 protein, S cerevisiae
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