Telomere length homeostasis

The physical ends of chromosomes, known as telomeres, protect chromosome ends from nucleolytic degradation and DNA repair activities. Conventional DNA replication enzymes lack the ability to fully replicate telomere ends. In addition, nucleolytic activities contribute to telomere erosion. Short telomeres trigger DNA damage checkpoints, which mediate cellular senescence. Telomere length homeostasis requires telomerase, a cellular reverse transcriptase, which uses an internal RNA moiety as a template for the synthesis of telomere repeats. Telomerase elongates the 3′ ends of chromosomes, whereas the complementary strand is filled in by conventional DNA polymerases. In humans, telomerase is ubiquitously expressed only during the first weeks of embryogenesis, and is subsequently downregulated in most cell types. Correct telomere length setting is crucial for long-term survival. The telomere length reserve must be sufficient to avoid premature cellular senescence and the acceleration of age-related disease. On the other side, telomere shortening suppresses tumor formation through limiting the replicative potential of cells. In recent years, novel insight into the regulation of telomerase at chromosome ends has increased our understanding on how telomere length homeostasis in telomerase-positive cells is achieved. Factors that recruit telomerase to telomeres in a cell cycle-dependent manner have been identified in Saccharomyces cerevisiae. In humans, telomerase assembles with telomeres during S phase of the cell cycle. Presumably through mediating formation of alternative telomere structures, telomere-binding proteins regulate telomerase activity in cis to favor preferential elongation of the shortest telomeres. Phosphoinositide 3-kinase related kinases are also required for telomerase activation at chromosome ends, at least in budding and fission yeast. In vivo analysis of telomere elongation kinetics shows that telomerase does not act on every telomere in each cell cycle but that it exhibits an increasing preference for telomeres as their lengths decline. This suggests a model in which telomeres switch between extendible and nonextendible states in a length-dependent manner. In this review we expand this model to incorporate the finding that telomerase levels also limit telomere length and we propose a second switch between a non-telomerase-associated "extendible” and a telomerase-associated "extending” stat

Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process

Mechanism and regulation of the nonsense-mediated decay pathway

The Nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs) but also regulates the abundance of a large number of cellular RNAs. The central role of NMD in the control of gene expression requires the existence of buffering mechanisms that tightly regulate the magnitude of this pathway. Here, we will focus on the mechanism of NMD with an emphasis on the role of RNA helicases in the transition from NMD complexes that recognize a PTC to those that promote mRNA decay. We will also review recent strategies aimed at uncovering novel trans-acting factors and their functional role in the NMD pathway. Finally, we will describe recent progress in the study of the physiological role of the NMD response

Saccharomyces cerevisiae Ebs1p is a putative ortholog of human Smg7 and promotes nonsense-mediated mRNA decay

The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast

A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon–intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease

A topological mechanism for TRF2-enhanced strand invasion

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