Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.
Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy.
Elena
E
Tosoni
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy.
Ilaria
I
Frasson
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA.
Matteo
M
Scalabrin
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy.
Rosalba
R
Perrone
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy.
Elena
E
Butovskaya
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy.
Matteo
M
Nadai
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy.
Giorgio
G
Palù
The RNA Institute, University at Albany-SUNY, Albany, NY 12222, USA.
Dan
D
Fabris
Department of Molecular Medicine, University of Padua, via Gabelli 63, 35121 Padua, Italy sara.richter@unipd.it.
Sara N
SN
Richter
D002460:Cell Line; D054856:G-Quadruplexes; D020868:Gene Silencing; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D000097796:Nucleolin
D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin