Widespread Translational Remodeling during Human Neuronal Differentiation

Blair, John D.; Hockemeyer, Dirk; Doudna, Jennifer A.; Bateup, Helen S.; Floor, Stephen N.

doi:10.1016/j.celrep.2017.10.095

Cited by 125 publications

(123 citation statements)

References 78 publications

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“…Alternatively, intronic sequences could act to recruit factors to inhibit translation, activate degradation during translation, or both. Analysis of the translation of splice variants with ribosome profiling found that intron retention events within the coding regions of transcripts were poorly translated (Weatheritt et al, 2016), consistent with our findings that transcripts with retained introns are often only bound by a single ribosome (Fig 6A) and other polysome fractionation studies (Floor & Doudna, 2016; Blair et al, 2017). Further study of the fate of transcript with retained introns will lead to a better understand of the true diversity of roles intron retention plays in gene expression.…”

Section: Discussionsupporting

confidence: 90%

“…Several models have been proposed to explain what marks a stop codon as authentic, where termination of translation occurs normally, versus a premature termination codon (PTC), which leads to NMD in animals, yeast and plants (Nagy & Maquat, 1998; Amrani et al, 2004; Bühler et al, 2006; Behm-Ansmant et al, 2007; Kérenyi et al, 2008; Singh et al, 2008; Hogg & Goff, 2010; Drechsel et al, 2013; Lloyd et al, 2018). Alternative splicing has a well known role in altering the coding potential of transcripts, but can also alter the translation efficiency of the transcript (Floor & Doudna, 2016; Weatheritt et al, 2016; Blair et al, 2017; Fagg et al, 2017; Reixachs-Sole et al, 2019) or change the stability of transcripts by targeting them to NMD (Jumaa & Nielsen, 1997; Hilleren & Parker, 1999; Lejeune et al, 2001; Lewis et al, 2003; Lareau et al, 2007; Ni et al, 2007; Floor & Doudna, 2016; Reixachs-Sole et al, 2019).…”

Section: Introductionmentioning

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: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

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

Lloyd

French

Brenner

2020

Preprint

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“…Using branched FISH, high-resolution confocal microscopy, and volumetric quantification of mRNA foci, we show that wild-type Htt mRNAs (with a normal number of CAG repeats) accumulate in the nucleus of neuronal cells, but not in non-neuronal cells. A similar Htt distribution pattern was demonstrated by the analysis of RNA sequencing (RNA-seq) datasets from human embryonic stem cell (HESC)-derived neurons (Blair et al, 2017; Figure S3E). The results showed that, relative to the total number of reads in each sample, Htt transcript is detected more often in the nuclear fraction than in the cytoplasmic fraction.…”

Section: Discussionsupporting

confidence: 72%

“…Processed nuclear and cytoplasmic RNA-seq datasets across neuronal differentiation from (Blair et al, 2017) were downloaded from the Gene Expression Omnibus (GSE100007) and TPM values for Htt mRNA (ENSG0000197386) were extracted for plotting.…”

Section: Quantification and Statistical Analysismentioning

confidence: 99%

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

Didiot

Ferguson

et al. 2018

Cell Reports

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SUMMARY Huntington’s disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ~50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA sub-cellular localization might play a role in important neuronal regulatory mechanisms.

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“…Binding sites (and their relative strengths) identified by these methods represent numerical averages over numerous mRNA isoforms, some of which might not even contain the actual binding sites. Isoform-specific association data are important because differential mRNP binding is thought to underpin many of the functional differences observed in same-gene isoforms, including variable translational efficiency, cellular localization, and stability (Blair, Hockemeyer, Doudna, Bateup, & Floor, 2017;Geisberg, Moqtaderi, Fan, Ozsolak, & Struhl, 2014). Alterations in distributions of isoforms whose sequences differ upstream of cleavage/polyadenylation sites (mRNA 3 isoforms) can contribute to misregulation of cell growth and differentiation and to increased oncogenic potential (Mayr, 2017).…”

Section: Introductionmentioning

confidence: 99%

Protein Binding to mRNA 3′ Isoforms

Geisberg

Moqtaderi

2019

CP Molecular Biology

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Here we describe CLIP‐READS, a technique that combines elements of crosslinking and immunoprecipitation (CLIP) and 3′ region extraction and deep sequencing (READS), to provide a genome‐wide map of mRNA 3′ isoform binding by a given messenger ribonucleoprotein (mRNP). In CLIP‐READS, cells are grown to logarithmic phase and are irradiated with UV light (254 nm) to form RNA–protein adducts. The protein−mRNA complexes are immunoprecipitated from cell extracts with an antibody specific to the protein of interest, after which the protein component is digested away with Pronase. Messenger RNAs are then subjected to 3′ READS. An input sample processed by 3′ READS in parallel allows for the relative quantification of isoform‐specific binding by the mRNP of interest. © 2019 by John Wiley & Sons, Inc.

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Widespread Translational Remodeling during Human Neuronal Differentiation

Cited by 125 publications

References 78 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

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

Protein Binding to mRNA 3′ Isoforms

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