Elsevier

Experimental Cell Research

Volume 313, Issue 20, 10 December 2007, Pages 4180-4195
Experimental Cell Research

Research Article
The Shwachman–Bodian–Diamond syndrome associated protein interacts with HsNip7 and its down-regulation affects gene expression at the transcriptional and translational levels

https://doi.org/10.1016/j.yexcr.2007.06.024Get rights and content

Abstract

The Shwachman–Bodian–Diamond syndrome (SDS) is an autosomal disorder with pleiotropic phenotypes including pancreatic, skeletal and bone marrow deficiencies and predisposition to hematological dysfunctions. SDS has been associated to mutations in the SBDS gene, encoding a highly conserved protein that was shown to function in ribosome biogenesis in yeast. In this work, we show that SBDS is found in complexes containing the human Nip7 ortholog. Analysis of pre-rRNA processing in a stable SBDS knock-down HEK293-derivative cell line revealed accumulation of a small RNA which is a further indication of SBDS involvement in rRNA biosynthesis. Global transcription and polysome-bound mRNA profiling revealed that SBDS knock-down affects expression of critical genes involved in brain development and function, bone morphogenesis, blood cell proliferation and differentiation, and cell adhesion. Expression of a group of growth and signal transduction factors and of DNA damage response genes is also affected. In SBDS knock-down cells, 34 mRNAs showed decreased and 55 mRNAs showed increased association to polysomes, among which is a group encoding proteins involved in alternative splicing and RNA modification. These results indicate that SBDS is required for accurate expression of genes important for proper brain, skeletal, and blood cell development.

Introduction

The Shwachman–Bodian–Diamond syndrome (SDS) is an autosomal recessive disease characterized by pleiotropic phenotypes including pancreatic insufficiency, hematological dysfunctions, and skeletal abnormalities. Other organs such as liver, kidneys, teeth, and the immune system may also be affected in SDS patients, which are predisposed to develop hematology abnormalities, including cytopenias, myelodysplasia, and leukemia [1], [2]. In 90% of the cases, SDS is associated to mutations in the SBDS gene. Recurring mutations as well as complex rearrangements in the SBDS gene arise from conversion events with an adjacent pseudogene, SBDSP, showing 97% sequence identity to the SBDS functional gene [3]. Human SBDS is a ubiquitously expressed nucleolar protein [4], member of a highly conserved protein family found in Archaea and eukaryotes. Knock-out of the yeast ortholog (Sdo1p) and double knock-out of the murine ortholog lead to lethality [5], [6]. The hypothesis of SBDS involvement in ribosome biogenesis came initially from archaeal orthologs, which are located within RNA processing operons that include orthologs of the eukaryotic exosome and RNase P complex subunits [7]. Subsequently, Sdo1p was found in complexes with ribosomal RNA (rRNA) processing and components of the 60S particle [5], [8]. A recent study based on yeast genetics strongly implicates Sdo1p in ribosome function by showing that it acts on the release and recycling of the ribosomal biogenesis factor Tif6 from pre-66S particles as well as on translation activation of ribosomes [9]. In addition, Sdo1p-deficient cells showed a reduced level of mature 60S subunits. Evidence for a SBDS role in RNA metabolism is provided also by the plant and trypanosomatid SBDS orthologs that contain a C-terminal extension containing RNA binding motifs [3].

In addition to SDS, other human genetic syndromes have been associated to loss-of-function mutations in genes encoding proteins involved in pre-rRNA processing and ribosome biogenesis, which include Diamond Blackfan Anemia (DBA), cartilage-hair hypoplasia (CHH), the Treacher Collins syndrome, and Dyskeratosis Congenita (DC) (reviewed by Liu and Ellis) [10]. Defects in structural components of mature ribosomes may cause phenotypes similar to those caused by defects in trans-acting factors required for pre-rRNA cleavage and covalent modification. The first gene described to be mutated in approximately 25% of DBA patients was RPS19, encoding the small ribosome subunit protein 19 [11]. Some authors have proposed that RPS19 may possess extra-ribosomal functions. Accordingly, RPS19 interacts with Pim1, linking the translational machinery to signal transduction pathways [12] and possibly also controlling gene expression at the translational level. CHH, on the other hand, has been linked to mutation in the RNA component of the RNase MRP that, among many functions in mitochondrial tRNA processing [13], is responsible for endonucleolytic cleavage of ITS1 and its efficiency affects 5.8S rRNA maturation and 60S subunit biogenesis [14]. The Treacher Collins associated nuclear phosphoprotein treacle interacts with RNA polymerase I and Nop56, a protein subunit of the box CD snoRNP, and has a role in rRNA methylation [15], [16]. Dyskeratosis congenita has been associated to mutations in the X-linked DKC1 gene [17], which encodes dyskerin, a pseudouridine synthase that in association with the box H/ACA snoRNP (including proteins Nop10, Gar1, and Nhp2) modifies ribosomal RNA [18]. Reconstitution studies have shown that deficiency in dyskerin abrogates pseudouridylation of rRNA causing a ribosome malfunction that affects translation of mRNAs containing internal ribosomal entry sites (IRES) [19], [20]. However, dyskerin, along with the other protein subunits of the box H/ACA snoRNP, has also been described as a component of the telomerase reverse transcriptase complex (TERT) [21]. The telomerase RNA component (TERC) contains a box H/ACA like domain and is responsible for attracting dyskerin to the telomerase complex. Shortened telomeres have been described both in DC [22] and in SDS patients [23], providing a further link between the two syndromes. The IRES translational defect caused by DKC1 knock-out [19] indicates that at least part of the phenotypes observed in DC seem to result from ribosome malfunction, affecting translation of a specific group of mRNAs and, consequently, expression of a restricted number of genes controlling cell growth and proliferation in the affected tissues and organs.

As described above, the yeast model system provided information on the basic function of the SBDS ortholog on 60S ribosome subunit maturation and on Tif6 recycling [9]. Studies using mouse as a model are hindered because the double knock-out Sbds(−/−) shows developmental arrest that leads to lethality whereas Sbds(+/−) mice have apparently normal phenotypes [6]. Therefore, we have established a human knock-down cell model using the small hairpin RNA interference (shRNA) technology to mimic SBDS deficiency. In this work, we describe the characterization of the shRNA knock-down cells, which accumulate a small pre-RNA precursor, and the interaction of SBDS with HsNip7, the human ortholog of yeast Nip7p, a protein required for 27S pre-rRNA cleavage and 60S subunit biogenesis [24]. We present also global transcription and polysome-bound mRNA profilings which revealed that SBDS knock-down affects expression of several groups of genes including genes involved in brain development and function, bone morphogenesis, blood cell proliferation and differentiation, cell adhesion, growth and signal transduction factors, and DNA damage response genes. In SBDS knock-down cells, only 34 mRNAs showed decreased association to polysomes whereas 55 mRNAs showed increased association, among which is a group encoding proteins involved in alternative splicing and RNA modification. A large fraction of the genes with altered expression control fundamental processes during development and cell homeostasis, providing insights into the gene expression alteration that may be responsible for causing SDS and its associated complications.

Section snippets

Plasmid construction and production of recombinant proteins

The SBDS cDNA was amplified by PCR from a human fetal brain cDNA library using primers ONZ299 and ONZ301 (Supplemental Table 1) and inserted into the NdeI–BamHI restriction sites of plasmid pET28a to generate pET-SBDS. The SBDS cDNA amplified by PCR using primers ONZ298 and ONZ300 (Supplemental Table 1) was subcloned into the EcoRI–SalI sites of pET-GST-TEV [25] and pGAD-C2, generating plasmids pET-GST-TEV-SBDS and pGAD-SBDS. The cDNA of human Nip7p was amplified by PCR using primers ONZ233 and

Analysis of SBDS interaction with HsNip7

Evidence such as the Nip7p requirement for the final steps of 60S subunits maturation and Sdo1p association to the pre-60S complex in yeast, in addition to the conservation of the ribosome biogenesis machinery, led us to investigate whether SBDS was able to interact with HsNip7. This interaction was first tested using the yeast two-hybrid system and both reporters of the two-hybrid assay were activated (Fig. 1A) at high levels, indicating an interaction between SBDS and HsNip7. The association

SBDS interaction with HsNip7 and its possible role in pre-rRNA processing

The yeast SBDS ortholog, Sdo1p, associates with a complex containing over 20 proteins involved in 60S subunit ribosome biogenesis [5] and has been implicated in the release and recycling of the ribosomal biogenesis factor Tif6 from pre-66S particles as well as on translation activation of ribosomes [9]. Sdop1 deficiency leads to an imbalance of the 60S/40S ribosome subunit ratio, consistent with an Sdo1p function in 60S ribosome maturation [9]. Nip7p is a conserved protein required for proper

Acknowledgments

We thank B. S. Fletcher (University of Florida) and C. A. Bonjardim (UFMG) for kindly providing the pMaleficent vector and HEK293 cells, respectively, Adriana C. Alves, Tereza C. Lima Silva, and Zildene G. Correa for technical assistance. This work was supported by FAPESP grants (CEPID/CBME 98/14138-2; SMolBNet 00/10266-8 and 06/02083-7). CH was a recipient of a fellowship from CNPq/MCT.

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