HALP, A Novel Transcriptional U Three Protein (t-UTP), Activates RNA Polymerase I Transcription by Binding and Acetylating the Upstream Binding Factor (UBF)

Qunhui Peng,Xiaojuan Du,Liangliang Zhang,Wei Han,Ruirui Kong,Lelin Hu,Yang Ke

doi:10.1074/jbc.m110.173393

Qunhui Peng, Xiaojuan Du + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.173393

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Abstract

Transcription of ribosome RNA precursor (pre-rRNA) and pre-rRNA processing are coordinated by a subset of U three proteins (UTPs) known as transcriptional UTPs (t-UTPs), which participate in pre-rRNA transcription in addition to participation in 18 S rRNA processing. However, the mechanism by which t-UTPs function in pre-rRNA transcription remains undetermined. In the present study, we identified hALP, a histone acetyl-transferase as a novel t-UTP. We first showed that hALP is nucleolar, and is associated with U3 snoRNA and required for 18 S rRNA processing. Moreover, depletion of hALP resulted in a decreased level of 47 S pre-rRNA. Ectopic expression of hALP activated the rDNA promoter luciferase reporter and knockdown of hALP inhibited the reporter. In addition, hALP bound rDNA. Taken together these data identify hALP as a novel t-UTP. Immunoprecipitation and GST pulldown experiments showed that hALP binds the upstream binding factor (UBF) in vivo and in vitro. It is of importance that hALP acetylated UBF depending on HAT in vivo, and hALP but not hALP (ΔHAT) facilitated the nuclear translocation of the RNA polymerase I (Pol I)-associated factor 53 (PAF53) from the cytoplasm and promoted the association of UBF with PAF53. Thus, we provide a mechanism in which a novel t-UTP activates Pol I transcription by binding and acetylating UBF.

Highlights

In eukaryotes the nucleolus is a compartment for ribosome biosynthesis, which includes transcription of the ribosomal RNA precursor,4 processing of pre-rRNA, and sequential assembly of ribosomal large and small subunits
The GFP-hALP deletion mutants containing the C terminus including deletion mutants GFPhALP-HATϩC, GFP-hALP-C, and GFP-hALP-⌬HAT localized in the nucleolus but the deletion mutant GFP-hALP-N localized in the cytoplasm indicating that the N terminus was not required for the nucleolar localization
Compared with GFP-hALP-N, GFP-hALP-NϩHAT localized in the nucleus suggesting that the HAT domain may mediate its localization in the nucleus

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and hALP siRNA—U2OS, H1299, and 293T cells were grown in DMEM medium supplemented with 10%. For silencing hALP expression, siRNA (small interference RNA) targeting hALP [44]: 5Ј-CAGCACCACUGCUGAGAAUAAGA-3Ј was chemically synthesized together with an unrelated control siRNA (5Ј-ACUACCGUUGUUAUAGGUG-3Ј) (Shanghai GenePharma Co., Ltd) These synthesized siRNAs were transfected into cells at a concentration of 100 nM with Lipofectamine 2000TM (Invitrogen) according to the manufacturer’s instruction. U2OS cell lysates were prepared and incubated with anti-hALP antibody or anti-acetyl-lysine antibodycoupled protein-A agarose beads (Amersham Biosciences). GST-hALP bound Flag-UBF or GST-UBF bound Flag-hALP proteins were detected by Western blotting probed with anti-Flag antibody. For Re-ChIP assays, reactions were immunoprecipitated with the anti-hALP antibody as previously described [50]. Complexes were eluted by incubation in 10 mM DTT at 37 °C for 30 min and diluted 50 times with dilution buffer, followed by a second immunoprecipitation with either anti-UBF antibody or mouse IgG as described [52]

RESULTS

DISCUSSION

Yang Ke