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UPF1 is required for gene expression in mitochondria and for the elimination of paternal mtDNA

ABSTRACT: UPF1 is a well-conserved RNA helicase in eukaryotes, involved in nuclear and cytoplasmic processes of gene expression. This study presents ChIP-seq evidence for its RNA-dependent association with mtDNA transcription sites in Drosophila S2 cells, and RNA-seq data indicating its requirement for the correct expression of mtDNA genes. UPF1 localisation in mitochondria was observed by immunostaining and GFP-tagging in different fly tissues and cell types. During spermatogenesis, depletion of UPF1 but not of other NMD factors, resulted in major defects in meiosis and cytokinesis, leading to complete sterility. Notably, the few spermatids produced show abnormalities in the formation of the nebenkern and, unlike the wild type, retain the mtDNA.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE266138 | GEO | 2024/05/10

REPOSITORIES: GEO

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UPF1 is required for gene expression in mitochondria and for the elimination of paternal mtDNA

Project description:UPF1 is required for gene expression in mitochondria and for the elimination of paternal mtDNA

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A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.

Project description:Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase UPF1. Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for PTC-containing reporter mRNAs when compared to their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1) binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD-target marker. p-UPF1 is enriched on NMD target 3'UTRs along with SMG5 and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with FRET experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3'UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3'UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.

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A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.

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POLDIP2 is required for mtDNA elimination during spermatogenesis to ensure maternal inheritance in Drosophila melanogaster

Project description:Maternal inheritance of mitochondrial DNA (mtDNA) is highly conserved in metazoans. While many species eliminate paternal mtDNA during late sperm development to foster maternal inheritance, the regulatory mechanisms governing this process remain elusive. Through a large-scale genetic screen in Drosophila, we identified 47 mutant lines exhibiting substantial retention of mtDNA in mature sperm. We mapped one line to Poldip2, a gene predominantly expressed in the testis. Disruption of Poldip2 led to pronounced mtDNA retention in mature sperm and subsequent paternal transmission to progeny. Further investigation via imaging, biochemical analyses and ChIP assays revealed that POLDIP2 is a mitochondrial matrix protein capable of binding to mtDNA. Moreover, we uncovered that CLPX, a key component of the major mitochondrial protease, binds to POLDIP2 to co-regulate mtDNA elimination in Drosophila spermatids. This study shed light on the mechanisms underlying mtDNA removal during spermatogenesis, underscoring the pivotal role of this process in safeguarding maternal inheritance.

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Phosphorylated UPF1 (p-UPF1) RIP-seq using mouse Neuro-2A (N2A) neuroblastoma cells

Project description:To identify high-confidence NMD targets in mouse N2A neuroblastoma cells, we used our established transcriptome-wide RNA sequencing (RNA-seq) methodologies. Through parallel analyses of RNA-seq upon UPF1-knockdown (KD) and RNA immunoprecipitation (RIP-seq) footprinting of p-UPF1-bound RNAs, we identified 1027 high-confidence neuronal NMD targets.

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UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency 2

Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

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Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (CLIP-Seq)

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Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (RIP-Seq)

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UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2

Project description:Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. In this work, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by the RNA-binding status of UPF1. Our findings rationalize a key event in the metazoan NMD quality control pathway and progress our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

2024-05-01 | PXD049474 | Pride

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