A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Shintaro Imamura,Ryan D Murphey,Eriko Koshimizu,Leonard I Zon,Caroline Erter Burns,Kenkichi Masutomi,Thomas M Roberts,Christina Raftopoulou,Yoichi Imai,Shuji Kishi,Sarantis Gagos,Peter E Bayliss,Jun-Ichi Hanai,Junzo Uchiyama

doi:10.1371/journal.pone.0003364

Abstract

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words)

Highlights

Telomerase is a ribonucleoprotein complex required for the synthesis of telomere terminal repeats
We show that zebrafish telomerase reverse transcriptase (zTERT) knockdown causes hypochromic anemia at the onset of circulation, and that this is accompanied by the impaired differentiation of blood cells and their eventual apoptotic cell death leading to a severe reduction of hematopoietic cells (‘pancytopenia’), during embryogenesis
Our results demonstrate for the first time that zTERT promotes the development of hematopoietic cells through a non-canonical mechanism that is independent of the authentic telomerase activity of telomerase reverse transcriptase (TERT) and the role of this enzyme in telomere lengthening

Summary

Introduction

Telomerase is a ribonucleoprotein complex required for the synthesis of telomere terminal repeats. The essential components required for this activity are telomerase reverse transcriptase (TERT), the catalytic component, and telomerase RNA (TR) (or TERC; telomerase RNA component) which is the template for DNA repeat synthesis [1,2]. The reverse transcriptase (RT) motifs are essential for the enzymatic activity of TERT in synthesizing telomere repeats and play an important role in nucleotide addition and processivity in concert with its C-terminal domain [8,9]. The RNA-binding domains of TERT interact with TR to facilitate the elongation of the telomere repeats via the catalytic activity of the RT domain [11,12,13,14,15] Both the TR binding and RT domains of TERT must act in concert for the synthesis of telomere repeats [13]

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Results

Conclusion