Translational Regulation of Gene Expression by Cytoplasmic Nucleophosmin in AML: A Global Evaluation of RNA's Differentially Recruited to Polyribosomes.

Abstract

Nucleophosmin (NPM) exon 12 mutations are found in 50–60 percent of acute myelogenous leukemia (AML) with normal karyotype, and the altered protein (termed NPMc) is relocalized from the nucleolus to the cytoplasm. Multiple functions have been attributed to NPM, and research by other investigators demonstrated that NPM serves a critical role in nucleolar-to-cytoplasmic shuttling of rRNA's and in ribosome biogenesis. An early indication of the role of NPM in hematopoietic neoplasia was its fusion to the ALK protein in patients with anaplastic large cell lymphoma (ALCL). We and others have demonstrated that the expression of NPM-ALK regulates mediators of translational initiation, including mTOR, S6K1, and 4E-BP1, and we showed that NPM-ALK expression significantly alters the partitioning of mRNA's to polyribosomes. Regulators of translational initiation can be rate-limiting for neoplasia in animal models, and we hypothesized that NPMc promotes leukemogenesis in a similar manner by altering the recruitment of specific mRNA's to polysomes and thereby altering their translation. To evaluate this hypothesis, we assessed the global profile of polysomal mRNA in cells engineered to express NPMc. We generated pTRE2-NPMc(A)-Ba/F3 murine hematopoietic cells which express NPMc under the control of a tetracycline-inducible promoter. These cells carry “variant A” NPMc, which contains an insertion of a TCTG tetranucleotide at positions 956 through 959 and is the most common NPMc variant in AML. Control cells contained the empty vector. We used these cell lines to compare in a global manner the partitioning of mRNA species to polysomes, which were isolated using sucrose gradient fractionation of post-nuclear supernatants. From this enriched polysomal material we purified RNA, and the global profile of mRNA in these fractions was assessed by using Affymetrix mouse genome microarrays. The data generated in this effort was analyzed using the GeneChip robust multichip analysis (GCRMA) method. Significance Analysis of Microarrays (SAM) and Linear Models for Microarray Data (Limma) were employed to identify significant differentially expressed genes. We identified 345 genes whose mRNA's exhibited altered polysomal partitioning, including 253 increased in cells with NPMc and 192 with decreased polysome association (FDR≤ 0.05). These translationally regulated mRNA's encode proteins that control transcription, post-translational modification of proteins, and proteins associated with hematopoietic neoplasia. One of these differentially recruited mRNA's encodes the Dub-1 protein, a ubiquitin peptidase that has been implicated previously in hematopoietic cell growth control, and the mRNA showed a 6-fold increase in polysome recruitment. Interestingly, in our previous evaluation comparing cells with NPM-ALK versus the kinase-deficient variant, there was a 50-fold increase in the partitioning of Dub-1 mRNA to polysomes. These findings suggest that alteration of Dub-1 expression through mRNA partitioning might be commonly utilized by both NPMc as well as NPM-ALK. Collectively, these findings suggest that concerted disruption of gene transcription and translation might broadly mediate the pathogenesis of hemotopoietic neoplasia.