RESUMEN
Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to characterize the composition and dynamics of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Upf1-23 (Upf1, Upf2, Upf3) and Upf1-decappingUpf1-decapping contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association with RNA was partially dependent on Upf1-23 components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through conserved, successive Upf1-23 and Upf1-decapping complexes. This model can be extended to accommodate steps that are missing in yeast, to serve for further mechanistic studies of NMD in eukaryotes.
Asunto(s)
Modelos Biológicos , Complejos Multiproteicos/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas/metabolismo , ARN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complejos Multiproteicos/genética , ARN Helicasas/genética , ARN de Hongos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.
Asunto(s)
ARN Helicasas/química , Transactivadores/química , Biocatálisis , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , ARN/metabolismo , ARN Helicasas/metabolismo , Transactivadores/metabolismoRESUMEN
Helicases are molecular engines which translocate along nucleic acids (NA) to unwind double-strands or remodel NA-protein complexes. While they have an essential role in genome structure and expression, the rules dictating their processivity remain elusive. Here, we developed single-molecule methods to investigate helicase binding lifetime on DNA. We found that UPF1, a highly processive helicase central to nonsense-mediated mRNA decay (NMD), tightly holds onto NA, allowing long lasting action. Conversely, the structurally similar IGHMBP2 helicase has a short residence time. UPF1 mutants with variable grip on DNA show that grip tightness dictates helicase residence time and processivity. In addition, we discovered via functional studies that a decrease in UPF1 grip impairs NMD efficiency in vivo. Finally, we propose a three-state model with bound, sliding and unbound molecular clips, that can accurately predict the modulation of helicase processivity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Ácidos Nucleicos/metabolismo , ARN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Humanos , Factores de TiempoRESUMEN
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.
Asunto(s)
Productos del Gen tax/metabolismo , Interacciones Huésped-Patógeno/fisiología , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas/metabolismo , Transactivadores/metabolismo , Adenosina Trifosfato/metabolismo , Productos del Gen tax/genética , Células HeLa/virología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Mutación , Dominios Proteicos , Transporte de Proteínas , ARN Helicasas/genética , Transactivadores/genéticaRESUMEN
Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Edición Génica , Meiosis , Centro Organizador de los Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Femenino , Ratones , Ratones Transgénicos , Oocitos/citología , Huso Acromático/genéticaRESUMEN
Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue.