Treacle recruits RNA polymerase I complex to the nucleolus that is independent of UBF

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Abstract

Mutations in treacle lead to Treacher Collins syndrome (TCS), an autosomal dominant disorder of craniofacial development. Treacle associates with upstream binding factor (UBF) to regulate rRNA gene (rDNA) transcription, but the precise mechanisms mediated by treacle remain elusive. Here we show that the central repeated domain of treacle binds with RNA polymerase I (Pol I), while that the treacle C-terminus is involved in rDNA promoter recognition and UBF recruitment. Knockdown of treacle resulted in dispersion of Pol I and UBF away from nucleolus, whereas interactions of treacle with Pol I and rDNA promoter were not disrupted by UBF depletion. These findings indicate that treacle, but not UBF, is essential for nucleolar recruitment of Pol I transcription complex. Furthermore, C-terminally truncated treacle, mimicking TCS-associated mutations, failed to target to the nucleolus, possibly causing loss-of-function in the mutant treacle. Our observations support that TCS results from haploinsufficiency of treacle.

Introduction

Treacher Collins syndrome (TCS) is a disorder of craniofacial development with autosomal dominant inheritance [1]. TCS is caused by mutations in TCOF1 gene, which encodes treacle. While only 40% of TCS cases have a familial history, 60% arise from denovo mutations [2]. The mutations documented in both cases include splicing mutations, insertion, deletion, as well as missense and nonsense mutations arisen throughout the gene. Majority of these mutations introduce a premature termination codon into TCOF1, resulting in truncated treacle or nonsense mediated mRNA degradation [3].

Treacle is a nucleolar phosphoprotein composed of N- and C-termini and a central repeated domain with alternating acidic and basic clusters [3]. It has homology with another nucleolar phosphoprotein, Nopp140, which is involved in the pre-rRNA transcription and processing, spatial organization of nucleolus, and normal body development [4], [5], [6], [7]. Treacle localizes to nucleolus in interphase and remains at the nucleolar organization regions (NORs) during mitosis [8]. Interaction of treacle and upstream binding factor (UBF) has been demonstrated and treacle depletion inhibits rRNA transcription [8]. Moreover, treacle can associate with NOP56, a component of the ribonucleoprotein methylation complex, to participate in the pre-rRNA methylation [9]. The carboxyl terminal region of treacle is responsible for nucleolar targeting and for interactions with UBF and NOP56 [8], [9], [10]. Since the majority of mutations identified in TCS generate C-terminally truncated proteins, loss-of-function mutations of treacle may be the mechanism underlying TCS pathogenesis.

The first step of ribosome biogenesis is the initiation of rRNA transcription that starts from the formation of pre-initiation complex composed by SL1 and UBF at the rRNA gene (rDNA) promoter for recruitment of RNA polymerase I (Pol I) complex [11]. UBF binding sites occur throughout the rDNA sequences, not restricted to the rDNA promoter, suggesting a more general role of UBF in organizing rDNA chromatin [12], [13]. Basal levels of rRNA transcription can be initiated by SL1 and Pol I in the in vitro reconstitution system, while transcription is enhanced in the presence of UBF [14]. SL1, independent of UBF, is able to associate with the rDNA promoter and conversely stabilizes UBF at the promoter. Our present study revealed that treacle, rather than UBF, was crucial for the maintenance of Pol I machinery in the nucleolus.

Section snippets

Materials and methods

Antibodies. Mouse monoclonal antibodies used included M2 (IgG1; anti-Flag; Sigma), F-9 (IgG1; anti-UBF; Santa Cruz), B-7 (IgG2a; anti-tubulin; Santa Cruz), BU33 (IgG1; anti-Br-dUTP; Sigma), CL17 (IgG1; anti-treacle), CL74 (IgG1, anti-treacle), KK1 (IgG2a; anti-treacle), CP2 (IgG2a; anti-hNopp140), and JY10 (IgG2a; anti-Pol I RPA 194). Rabbit polyclonal antibodies used included F7425 (anti-Flag; Sigma), H-300 (anti-UBF; Santa Cruz), and H-140 (anti-fibrillarin; Santa Cruz).

Mammalian expression

Treacle associates with Pol I and hNopp140 (human Nopp140)

To investigate the hNopp140-associated proteins, we generated monoclonal antibodies to cell components precipitated by anti-hNopp140 antibody and isolated the corresponding cDNAs from expression libraries by immunoscreening. One of the proteins was identified as treacle. Double immunostaining with anti-treacle and anti-hNopp140 antibodies revealed that treacle appeared as small dots colocalized with hNopp140 in interphase nucleoli (Fig. 1A, arrow). In M phase cells, hNopp140 dispersed into

Discussion

Ribosome biogenesis, accounting for a large proportion of metabolic effort of a cell, is tightly regulated during development for the demands of individual cells when proliferation, apoptosis, and cell fate decision take place. Recent studies have shown that rRNA gene silencing regulated through reduced UBF expression is coordinated to cell differentiation [18]. Knockdown of Nopp140 induces minute-like phenotypes, characterized by wing, leg, and tergite deformities in Drosophila[6]. Since

Acknowledgments

This research was supported by grants from the National Science Council (NSC 95-2320-B-010-057-MY3) and the National Health Research Institutes (NHRI-EX94-9003BL).

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