NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements

Abstract

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

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

“…Tight control of NMD factor concentrations through autoregulation buffers such undesired consequences. Previously, individual NMD factor-encoding mRNAs have already been reported to be targeted by NMD in different organisms: SMG5 has been identified as an NMD target in human and fruit fly cells (Mendell et al 2004;Rehwinkel et al 2005;Chan et al 2007), SMG7 and UPF3 in plants (Kerenyi et al 2008;Saul et al 2009), SMG6 in fruit fly (Rehwinkel et al 2005), and UPF1 in mouse (Weischenfeldt et al 2008). Thus the selfregulation of NMD that we demonstrated in this study for human cells likely extends to other organisms.…”

Introns in UTRs: Why we should stop ignoring them

“…In addition to facilitating elimination of the nonfunctional mRNAs described above, significant evidence exists that some 3UIs serve to modulate normal gene expression [40,[43][44][45][46][47][48][49]. Three classes of 3UI-containing mRNAs are both functional and subject to NMD (Fig.…”

“…Insights & Perspectives ..... complete embryogenesis, with the most prominent defects observed in heart, brain, and hematopoietic development [45,47,73,74]. The neural development program incorporates NMD in at least two important ways, both with widespread effects.…”

“…As a result, hundreds of transcripts that would otherwise be subject to NMD are up-regulated, and most of these encode proteins important for neural function [46]. 3UI-containing transcripts are also enriched for mRNAs involved in amino acid metabolism, starvation, ER stress, and hypoxia [40,47,49]. These enrichments led to the discovery that NMD is inhibited during stress through a mechanism involving phosphorylation of translation initiation factor, eIF2a.…”

Essential Role of p400/mDomino Chromatin-remodeling ATPase in Bone Marrow Hematopoiesis and Cell-cycle Progression

“…The flow cytometric characterization of bone marrow hematopoietic cells was performed as described before (29).…”