C-terminal regions of the human telomerase catalytic subunit essential for in vivo enzyme activity

Abstract

Most human cancer cells are thought to acquire the ability to divide beyond the capacity of normal somatic cells through illegitimately activating the gene hTERT, which encodes the catalytic subunit of telomerase. While telomerase reverse transcriptase (TERT) is conserved in most eukaryotes, mounting evidence suggests that the C terminus of the human protein may have functions unique to higher eukaryotes. To search for domains responsible for such functions, we assayed a panel of tandem substitution mutations encompassing this region of human TERT for in vitro and in vivo functionality. We found four clusters of mutations that inactivated the biochemical and biological functions of telomerase, separated by mutations that had little or no effect on enzyme activity. We also identified a region where mutations generate catalytically active but biologically inert proteins. This C-terminal region that dissociates activities of telomerase (C-DAT) does not appear to be involved in nuclear localization or protein multimerization. Instead, it appears that the C-DAT region is involved in a step of in vivo telomere synthesis after the assembly of a catalytically active enzyme. Intriguingly, all of the described regions reside in a portion of TERT that is dispensable for cellular viability in yeast, arguing for a divergent role of the C terminus in higher eukaryotes.

FIG. 1.

NAAIRS substitution mutagenesis of the C terminus of hTERT identifies five regions essential for enzyme function. (A) Schematic representation of the position and effect on telomerase activity of the NAAIRS substitution mutations of hTERT. The C-terminal amino acid sequences of the human (h), mouse (m), and Arabidopsis (At) TERT protein are aligned. Boxed amino acids denote identical sequence between all three proteins. RT motifs 6 (or D) and 7 (or E), the putative NES and 14-3-3 binding site are denoted with a line. Highly conserved or criticalamino acids of these regions are shown as letters above the alignment. Sequences are divided into six amino acid segments corresponding to the positions of the NAAIRS substitutions in the hTERT protein. Numbers refer to the first amino acid of each substitution, starting with +1 as the initiating Methionine. The six amino acid segments are shaded in dark grey, medium grey, or light grey or are left unshaded to represent high, medium, low and undetectable levels of in vitro telomerase enzyme activity as defined in the text. Large boxes identify novel essential regions E-I, -II, -III, and -IV. (B) NAAIRS mutations with different levels of telomerase activity. Cell lysates (0.2 μg or threefold serial dilutions thereof) prepared from HA5 cells stably infected with retroviruses encoding the wild-type FLAG-hTERT or FLAG-hTERT NAAIRS mutants +1022, +920, +1118, and +1088 were assayed for telomerase activity. HT, heat treated. IS, the internal standard, is shown as a different exposure to more easily visualize the standard.

FIG. 2.

NAAIRS substitution in RT motifs 6 (or D) and 7 (or E), but not the spacer region, result in a protein that is catalytically inactive in vitro. (A) Alignment of the region encompassing the first eight NAAIRS substitutions between hTERT and mouse TERT (mTERT). Dissimilar amino acids are shaded. The approximate boundaries of RT motif 6 (or D) and motif 7 (or E) are denoted with a line. Highly conserved amino acids are highlighted in bold letters above the alignment. The alignment is shown in blocks of six amino acids, which correspond to the positions of the first eight NAAIRS substitutions. Numbers refer to the first amino acid of each substitution (+1 being the initiating methionine). (B) Cell lysate (0.2 μg) prepared from HA5 cells stably infected with the retroviral vector encoding FLAG-hTERT, no inserted gene (vector), or the FLAG-hTERT NAAIRS substitution mutants shown was assayed for telomerase activity. HT, heat treated; IS, internal standard.

FIG. 3.

Class one mutants are biochemically and biologically inactive. (A) C-terminal regions E-I, -II, -III, and -IV are essential for telomere maintenance. Telomere length of DNA isolated from cells expressing FLAG-hTERT, or NAAIRS mutants +998, +1034, +1088, or +1118 representing each of the four regions E-I, -II, -III, and -IV, was determined by Southern hybridization with a telomeric probe.(B) HA5 cells expressing hTERT containing a NAAIRS mutation in one of the four essential regions are mortal. Cell divisions in pds versus time in days is plotted for HA5 cells infected with FLAG-hTERT (▪), an empty vector (•), or the aforementioned representative FLAG-hTERT-NAAIRS mutants for E regions I (○), II (□), III (▵), and IV (⋄). (C) Mutations in regions E-I, -II, -III, and -IV do not alter RNA or protein levels of hTERT. Total RNA prepared from the same aforementioned cell lines, or the control HA5 cells stably expressing untagged hTERT, was RT-PCR amplified with primers specific for hTERT or GAPDH. Protein lysates prepared from 293 cells transiently transfected with an empty (vector) mammalian expression vector or one encoding FLAG-hTERT, hTERT, or representative FLAG-hTERT NAAIRS mutants for each of the four essential regions were resolved by SDS-PAGE and Western blotted with an anti-FLAG or antiactin antibody.

FIG. 4.

Mutations to essential regions I to IV do not affect hTR RNA binding. (A) hTR binding to hTERT E-region mutants. The ³⁵S-labeled FLAG-hTERT-FLAG protein and variants containing mutations at positions +998, +1034, +1088, and +1118, as well as the HDAC1-FLAG recombinant protein, were produced in the presence of ³²P-labeled hTR and immunoprecipitated with an anti-FLAG antibody. The hTR input lane represents 1/100th of the RNA remaining after immunoprecipitation. (B) FLAG-tagged hTERT is specifically immunoprecipitated. FLAG-hTERT-FLAG and hTERT-HA proteins were immunoprecipitated (IP) with the anti-FLAG antibody. Due to the poor expression of the hTERT-HA construct, the in vitro transcription and translation reaction was scaled up eightfold. Input lanes represent 1/20th of the total protein remaining after immunoprecipitation.(C) hTR-hTERT interactions are specific. Wild-type and hTR binding domain-defective hTERT proteins were synthesized in the presence of hTR or U6 RNA and immunoprecipitated. Input RNA lanes represent 1/100th of the total RNA added to the transcription-and-translation reaction. To more clearly differentiate protein and RNA components, the ³⁵S protein signal has been filtered out in the upper panel by the use of a blocking screen. Immunoprecipitated proteins are shown in the lower panel.

FIG. 5.

Telomerase activity detected in cells expressing class two hTERT mutants is not due to activation of the endogenous hTERT gene. Total RNA was prepared from each of the nine cell lines that had telomerase activity and divided beyond crisis, as well as from vector- and FLAG-hTERT-infected cells and the telomerase-positive cell line CWR22. This RNA was RT-PCR amplified with primers specific for total hTERT (endogenous or ectopic transcripts), endogenous hTERT, or, as a control, GAPDH mRNA. All but samples +914, +920, and +1052 were isolated at late passage.

FIG. 6.

Mutations to the +1127 C-DAT region render hTERT catalytically active but biologically inert.(A) HA5 cells expressing hTERT +1127 NAAIRS mutant are mortal. Cell division in pds versus time in days is plotted for HA5 cells infected with FLAG-hTERT (▪), an empty vector (•), or the aforementioned FLAG-hTERT-NAAIRS mutants +1127 (○), and +920 (□). (B) Cells expressing the +1127 NAAIRS mutant have shortened telomeres. DNA was isolated from cells expressing FLAG-hTERT, vector control, or the hTERT +1127 NAAIRS mutant, all at pd 9. Digested DNA was resolved and subject to Southern hybridization with a telomeric probe. (C) Cells expressing the hTERT +1127 NAAIRS mutant demonstrate chromosome instability near terminal passage. Representative metaphase spread is depicted with arrows indicating dicentric chromosomes. (D) The NAAIRS substitution at position +1127 in the C-DAT region does not drastically lower telomerase activity. Cell lysates (0.2 μg) prepared from HA5 cells stably infected with retroviruses encoding the wild-type FLAG-hTERT or an empty vector or FLAG-hTERT NAAIRS mutants +1127 or +920 were assayed for telomerase activity. HT, heat treated. IS, internal standard. Water was included as a negative control.(E) Catalytic activity of hTERT +1127 NAAIRS mutant is of a level sufficient for cellular immortalization. Cell lysates (0.2 μg and threefold serial dilutions thereof) were prepared from precrisis and postcrisis HA1 cells and HA1 cells stably infected with FLAG-hTERT+1127 NAAIRS and assayed for telomerase activity. HT, heat treated. IS, internal standard.

FIG. 7.

The sequence F₁₁₂₇KT of hTERT defines the biologically important C-DAT region. (A) Cell lysates (0.2 μg) were prepared from HA5 cells that were stably infected with FLAG-hTERT, vector or hTERT variants with mutations in the +1127 region, and assayed for telomerase activity. HT, heat treated. IS, internal standard. (B) The sequence F₁₁₂₇KT of hTERT is necessary for immortalization of HA5 cells. The last nine amino acids of the various versions of FLAG-hTERT are shown beside the life span of cells expressing these proteins. Amino acids in bold denote changes to primary sequence. The grey box highlights the region necessary for an immortal life span. EL, extended life span.

FIG. 8.

The +1127 C-DAT region does not appear to be involved in nuclear localization or hTERT multimerization. (A) Mutating the C-DAT region does not perturb the nuclear localization of hTERT. Human U2OS cells were transiently transfected with either YFP, YFP-hTERT, or variants of YFP-hTERT with NAAIRS substitutions in the 14-3-3 binding domain (+1034 NAAIRS) or the C-DAT region (+1127 NAAIRS). Representative cells expressing the aforementioned proteins were viewed as fluorescence images and photographed at a magnification of ×400. (B) Mutating the C-DAT region does not affect hTERT multimerization. FLAG- and GST-tagged proteins were expressed and ³⁵S labeled in vitro, mixed together, further incubated, and immunoprecipitated with an anti-FLAG antibody. hTERT proteins were expressed in wild-type form or with mutations in the C-DAT region. The irrelevant FLAG-tagged protein HDAC1 used as a negative control failed to immunoprecipitate GST-hTERT.