Download NFX1-123 Increases HTERT Post-Transcriptionally in HPV 16E6

NFX1-123 Increases HTERT Post-Transcriptionally in HPV 16E6-Keratinocytes
R Katzenellenbogen, FHCRC/University of Washington, Seattle, USA
P Vliet, University of Washington, Seattle, USA
D Galloway, FHCRC, Seattle, USA
Background: E6 induces telomerase activity through upregulation of hTERT, the
catalytic subunit of telomerase. Full activation of hTERT by 16E6 in keratinocytes
(HFKs) requires the endogenous protein NFX1-123. NFX1-123 contains a PAM2 motif,
to which cytoplasmic poly(A) binding proteins (PABPCs) bind, and an R3H domain, to
which singlestranded nucleic acids putatively can bind. The mechanism by which NFX1123 affects 16E6 activation of hTERT is unknown. Objective: To determine whether
NFX1-123 affects hTERT at the transcriptional or post-transcriptional level and the
domains required for this effect. Methods: HFKs expressing tagged (F-) NFX1-123, with
or without 16E6, were stained for immunofluorescence. 293T cells transfected with F
NFX1-123 were collected for immunoblot as well as treated with leptomycin B and
stained for immunofluorescence. HFKs expressing 16E6 and either wildtype F-NFX1123 or mutant F-NFX1-123, with the PAM2 motif or R3H domain deleted, were
collected for qPCR and telomeric repeat amplification protocol. Luciferase RNA fused
with the 5’ untranslated region (5’UTR) of hTERT or beta-actin was transfected into
HFKs expressing wildtype F-NFX1-123 or mutant F-NFX1-123, with or without 16E6,
and protein was collected. Results: NFX1-123 was cytoplasmic and unlike PABPCs, did
not shuttle between the cytoplasm and nucleus. In 16E6 FNFX1-123 HFKs, the two to
three fold increase in hTERT mRNA and telomerase activity required both the PAM2
motif and R3H domain of F-NFX1-123. Three fold more luciferase was produced in
16E6 F-NFX1-123 HFKs when luciferase RNA was fused with the 5’UTR of hTERT.
This post-transcriptional increase in expression required the PAM2 motif and R3H
domain of F-NFX1-123 and co-expression of 16E6. Conclusions: NFX1-123 is a
cytoplasmic protein that increases the hTERT mRNA and telomerase activity in 16E6
HFKs post-transcriptionally through its PAM2 motif and R3H domain.