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

Abstract

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.

ACKNOWLEDGMENTS

We gratefully acknowledge Joe Baur for his contributions to the development of a human extract-based assembly system for telomerase.

This work was supported by research grants from the National Institute on Aging (AG07992) and Geron Corporation. Postdoctoral support was from National Institutes of Health oncology training grant T32-CA66187 to L.P.F., National Institute on Aging grant AG05747 to S.E.H., and National Institutes of Health oncology training grant T32-CA66187 and an American Cancer Society postdoctoral fellowship to V.M.T. J.W.S. is an Ellison Medical Foundation Senior Scholar.