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Global analysis of Upf1 in mESCs reveals expanded scope of nonsense-mediated mRNA decay

ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a conserved RNA surveillance pathway that is an important modulator of disease pathology and is required for embryonic development. Despite significant research effort, the rules that govern NMD remain incompletely understood. Here we used a combined¬ approach, integrating RNA-Seq, ribosome footprinting, and CLIP-Seq analysis of the essential NMD factor Upf1, to provide a more complete picture of the role of NMD in modulating gene expression in murine embryonic stem cells (mESCs). We show that presence of an exon-exon junction ≥50 nucleotides (nt) downstream of a termination codon (dEJ) contributes to NMD independently of 3' UTR length, but has stronger effects in genes with shorter 3' UTRs. We also map translated upstream open reading frames (uORFs) in mESCs and show that they are associated with NMD regulation, especially of genes encoding transcription factors, and we find that lowly translated mRNAs can escape NMD. Finally, we identify over 200 direct binding targets of Upf1 and describe a pathway of Upf1-dependent gene regulation reliant on Upf1 binding to the 3' UTR and independent of presence of a dEJ. Together, these analyses characterize known and discover novel determinants of NMD and establish a broader role in mESC gene regulation for Upf1.

ORGANISM(S): Mus musculus

PROVIDER: GSE41785 | GEO | 2013/06/12

SECONDARY ACCESSION(S): PRJNA178171

REPOSITORIES: GEO

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Global analysis of Upf1 in mESCs reveals expanded scope of nonsense-mediated mRNA decay

Project description:Nonsense-mediated mRNA decay (NMD) is a conserved RNA surveillance pathway that is an important modulator of disease pathology and is required for embryonic development. Despite significant research effort, the rules that govern NMD remain incompletely understood. Here we used a combined¬ approach, integrating RNA-Seq, ribosome footprinting, and CLIP-Seq analysis of the essential NMD factor Upf1, to provide a more complete picture of the role of NMD in modulating gene expression in murine embryonic stem cells (mESCs). We show that presence of an exon-exon junction ?50 nucleotides (nt) downstream of a termination codon (dEJ) contributes to NMD independently of 3' UTR length, but has stronger effects in genes with shorter 3' UTRs. We also map translated upstream open reading frames (uORFs) in mESCs and show that they are associated with NMD regulation, especially of genes encoding transcription factors, and we find that lowly translated mRNAs can escape NMD. Finally, we identify over 200 direct binding targets of Upf1 and describe a pathway of Upf1-dependent gene regulation reliant on Upf1 binding to the 3' UTR and independent of presence of a dEJ. Together, these analyses characterize known and discover novel determinants of NMD and establish a broader role in mESC gene regulation for Upf1. mRNA-Seq analysis of wildtype (2 samples), translationally inhibited (by cycloheximide treatment, 2 samples), control-depleted (2 samples), and Upf1-depleted (4 samples) mouse embryonic stem cells (mESCs); CLIP-Seq analysis of Upf1 (5 samples, and 5 samples of IgG control CLIP-Seq); Ribosome footprint profiling of wildtype (1 sample), control-depleted (1 sample), and Upf1-depleted (1 sample) mESCs

2013-06-12 | E-GEOD-41785 | biostudies-arrayexpress

Divergent effects of eRF3 and Upf1 on the expression of uORF carrying mRNAs and ribosome protein genes

Project description:In addition to its role in translation termination, eRF3 has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with Upf1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbor upstream open reading frames in their 5' leader sequence and long 3′ UTR. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3 or Upf1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3 and Upf1 and to compare the effect of each of these factors on gene expression. We show that eRF3 and Upf1 have very different impact on the human transcriptome, only ~250 transcripts being targeted by both factors. We also show that in contrast to Upf1, eRF3a depletion globally derepressed the expression of mRNAs containing translated uORFs. Finally, we find that eRF3a and Upf1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.

2019-10-27 | GSE126385 | GEO

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. RIP-seq experiments for p-UPF1, control IPs using rabbit IgG and additional control sample without IP were performed in duplicates

2014-08-15 | E-GEOD-60045 | biostudies-arrayexpress

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

2014-08-15 | GSE60045 | GEO

TDRD6 instructs correct assembly of mRNPs in the chromatoid body to potentiate UPF1 dependent mRNA decay.

Project description:RNA sequencing of heterozygote or Tudor domain contian protein 6 (TDRD6) knockout round spermatid cells. Chromatoid bodies (CBs) are germ cell-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family such as TDRD6. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay machinery such as UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 interaction, and for assembly of UPFs and other RNA binding proteins into super-complexes. In absence of TDRD6, the association of some mRNAs with UPF1 is impaired, and the long 3’ UTR-stimulated but not the exon junction complex-stimulated pathway of NMD is distorted. Reduced association of mRNAs with UPF1 correlated with increased stability and presence in polysome fractions, i.e. enhanced translational activity. Thus, we define CBs as sites of UPF1-dependent mRNA degradation and provide evidence for the requirement for NMD in spermiogenesis. This function of CBs depends on TDRD6-promoted assembly of mRNA decay enzymes within mRNPs.

2015-12-08 | GSE63948 | GEO

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

Project description:RNA quality control pathways serve to get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the ATPase cycle of SF1 Helicase Upf1 is required for mRNA discrimination during Nonsense-Mediated Decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD - translation and termination codon-proximal poly(A) binding protein - depend on Upf1 ATPase to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase-dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be an underappreciated, yet widely employed, activity in target RNA discrimination. CLIP and RIP-seq against Wild Type and Mutant Upf1 in HEK293-T cell lines

2015-07-06 | E-GEOD-69584 | biostudies-arrayexpress

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

2015-07-06 | E-GEOD-69585 | biostudies-arrayexpress

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

2015-07-06 | GSE69584 | GEO

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

2015-07-06 | GSE69585 | GEO

Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3’ UTRs

Project description:The RNA helicase UPF1 is best known for its key function in mRNA nonsense-mediated mRNA decay (NMD), but has also been implicated in additional mRNA turnover mechanisms, telomere homeostasis, and DNA replication. In NMD, UPF1 recruitment to target mRNAs is thought to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3’ untranslated region (UTR) of mRNAs has also been reported. To map UPF1 binding sites transcriptome-wide, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation by puromycin. We found a strong association of UPF1 with 3’ UTRs in undisturbed, translationally active cells and a significant increase in UPF1 binding to coding sequence (CDS) after translation inhibition. These results indicate that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This evidence for translation-independent UPF1-RNA interaction, which is corroborated by RNA immunoprecipitations experiments and by our observation that UPF1 also crosslinks to long non-coding RNAs, suggests that the decision to trigger NMD occurs after association of UPF1 with the mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination.

2013-06-20 | GSE47976 | GEO

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