Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay

Hoek, Tim A.; Khuperkar, Deepak; Lindeboom, Rik G.H.; Sonneveld, Stijn; Verhagen, Bram M.P.; Boersma, Sanne; Vermeulen, Michiel; Tanenbaum, Marvin E.

doi:10.1016/j.molcel.2019.05.008

Cited by 133 publications

(149 citation statements)

References 54 publications

(88 reference statements)

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“…NMD is a pervasive mechanism, influencing the expression of thousands of genes in humans (Mendell et al, 2004; Colombo et al, 2017); however, it is not a trivial matter to determine which transcripts are direct NMD targets and what features of a transcript trigger NMD. Studying the steady-state expression of transcripts after NMD inhibition has shown that exon-exon junctions located ≥50 nt downstream of a stop codon are potent triggers of NMD, while it is less clear as to what extent long 3’ UTRs act to trigger NMD (Lindeboom et al, 2016; Colombo et al, 2017; Tian et al, 2017; Lloyd et al, 2018; Hoek et al, 2019). By examining mutations in tumors and the corresponding changes in expression of these genes, it was shown that the EJC model was able to account for the majority of expression changes in mutations leading to early stop codons, and found that the length of the 3’ UTR was not a factor (Lindeboom et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…By examining mutations in tumors and the corresponding changes in expression of these genes, it was shown that the EJC model was able to account for the majority of expression changes in mutations leading to early stop codons, and found that the length of the 3’ UTR was not a factor (Lindeboom et al, 2016). In single molecule studies of transcripts, exon-exon junctions were found to be a potent trigger of NMD while the long 3’ UTRs tested were not (Hoek et al, 2019). In this study, we used the abundance of transcripts in the different polysome fractions to explore the features of transcripts linked to NMD.…”

Section: Discussionmentioning

confidence: 99%

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Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

Lloyd

French

Brenner

2020

Preprint

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Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA surveillance pathway that eliminates transcripts with premature termination codons. Several studies have tried defined the features governing which transcripts are targeted to NMD. However, these approaches often rely on inhibiting core NMD factors, which often have roles in non-NMD processes within the cell. Based on reports that NMD-targeted transcripts are often bound by a single ribosome, we analyzed RNA-Seq data from a polysome fractionation experiment (TrlP-Seq) to characterize the features of NMD-targeted transcripts in human cells. This approach alleviates the need to inhibit the NMD pathway. We found that the exon junction complex (EJC) model, wherein an exon-exon junction located ≥50 nucleotides downstream of a stop codon is predicted to elicit NMD, was a powerful predictor of transcripts with high abundance in the monosome fraction (bound by a single ribosome). This was also true for the presence of an upstream open reading frame. In contrast, as 3’ UTR lengths increase, the proportion of transcripts that are most abundant in the monosome fraction does not increase. This suggests that either longer 3’ UTRs do not consistently act as potent triggers of NMD or that the degradation of these transcripts is mechanistically different to other NMD-targeted transcripts. Of the ribosome-associated transcripts annotated as “non-coding”, we find that a majority are bound by a single ribosome. Many of these transcripts increase in response to NMD inhibition, including the oncogenic SHNG15, suggesting many might be NMD targets. Finally, we found that retained intron transcripts without a premature termination codon are over-represented in the monosome fraction, suggesting an alternative mechanism is responsible for the low level of translation of these transcripts. In summary, our analysis finds that the EJC model is a powerful predictor of NMD-targeted transcripts, while the presence of a long 3’ UTR is not.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

Lloyd

French

Brenner

2020

Preprint

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“…To measure the fluorescence intensities over time on single mRNAs, we used a Matlab-based software package called 'TransTrack' that we have recently developed (Boersma et al, 2019). Using TransTrack, we tracked single mRNAs from the start of translation, measured the GFP fluorescence intensity of each 48 translation site and performed local background subtraction.…”

Section: Arrival Time Of the First Ribosome At The Sirna Binding Sitementioning

confidence: 99%

“…Because translation termination of a single ribosome cannot be readily distinguished from mRNA cleavage, as they both result in the separation of a single ribosome/SunTag array from the mRNA, we used mRNA disappearance (i.e. decay of the 3' cleavage fragment) as a read-out for mRNA cleavage (Hoek et al, 2019). Therefore, we determined the time from GFP appearance to mRNA disappearance for each mRNA molecule.…”

Section: Analysis Of Mrna Cleavage With the Emi1-kif18b Reportermentioning

confidence: 99%

mRNA structural dynamics shape Argonaute-target interactions

Ruijtenberg

Sonneveld

Cui

et al. 2019

Preprint

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Small RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions.Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

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“…An important feature of NMD is that it is dependent on translation, either on the first or a later round [9][10][11][12] . Evidence supporting the formation of complexes containing NMD and translation termination factors suggested a functional connection between the two processes [13][14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

Human NMD ensues independently of stable ribosome stalling

Karousis

Gurzeler

Annibaldis

et al. 2019

Preprint

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Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathwaythat is important for the elimination of faulty and the regulation of normal mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system revealed similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs.Moreover, ribosome profiling revealed a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

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Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay

Cited by 133 publications

References 54 publications

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

mRNA structural dynamics shape Argonaute-target interactions

Human NMD ensues independently of stable ribosome stalling

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