Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival.[Keywords: Hematopoietic stem and progenitor cells; T-cell development; nonsense-mediated mRNA decay; programmed DNA rearrangements; alternative splicing; pseudogenes] Supplemental material is available at http://www.genesdev.org. Nonsense-mediated mRNA decay (NMD) is part of a larger network of RNA surveillance pathways, which ensure that only mRNAs with proper coding potential become available for protein synthesis. Specifically, the NMD machinery recognizes mRNAs with premature termination codons (PTCs) and mediates their degradation. If left available for translation, PTC-containing (PTC + ) mRNAs pose a latent threat to the cell as they may encode proteins with potential dominant-negative properties.The NMD pathway was initially considered as a guardian against newly arising nonsense mutations either at the DNA level or as a consequence of erroneous transcription or mRNA processing (for recent reviews, see Conti and Izaurralde 2005;Isken and Maquat 2007). More recently, global expression profiling of tissue culture cells subjected to siRNA-mediated knockdown of key NMD components demonstrated that even nonmutated genes are regulated by NMD, suggesting that the NMD pathway actively participates in regulation of normal gene expression (Mendell et al. 2004;Rehwinkel et al. 2005;Wittmann et al. 2006). Of particular importance are the T-cell receptor (TCR) and immunoglobulin (Ig) genes, which undergo programmed DNA rearrangements. Here, two-thirds of the V(D)J recombination events are predicted to be nonproductive by generating a PTC, and the resulting PTC + transcripts were shown to be stabilized both by inhibition of protein synthesis and

BackgroundNonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2.ResultsWe developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation.ConclusionsOur data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.

CCAAT/enhancer binding protein (C/EBP)α is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which dissociate the ability of C/EBPα to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations—all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count—normally associated with AML—were absent. These results show that disrupting the cell cycle regulatory function of C/EBPα is sufficient to initiate AML-like transformation of the granulocytic lineage, but only partially the peripheral pathology of AML.

The management of disordered calcium and phosphate homeostasis in CKD patients is evolving based on our knowledge of the major importance of the calcium sensing receptor (CASR) in controlling parathyroid gland function and the potent actions of calcimimetics to target CASR. The purpose of this presentation is to provide an overview of the role of the CASR in regulation of parathyroid gland function, to examine the mechanisms whereby calcimimetics target the CASR, and to review the clinical trials that support the use of cinacalcet HCl for the treatment of secondary hyperparathyroidism in stage 5 chronic kidney disease (CKD).