Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress

  1. Showkat A. Dar
  2. Sulochan Malla
  3. Vlastimil Martinek
  4. Matthew J. Payea
  5. Christopher T. Lee
  6. Jessica Martin
  7. Aditya J. Khandeshi
  8. Jennifer L. Martindale
  9. Cedric Belair
  10. Manolis Maragkakis

  • Curated by eLife

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    eLife assessment

    This valuable study describes mRNA shortening during cellular stress and interestingly observes that this shortening is dependent on localization in stress granules. Surprisingly, this mRNA shortening does not appear to require the shortening of polyA tails. These are in principle novel findings, but the evidence for them is currently incomplete. Additional experiments would help bolster confidence in how the authors interpret their data.

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Abstract

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5’ end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5’ end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on removal of the poly(A) tail. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome. Inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 fully rescues RNA length and suppresses stress-induced decay. Our findings reveal RNA decay as a key determinant of RNA metabolism upon cellular stress and dependent on stress-granule formation.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/11002926.

    The manuscript "Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress" by Dar and colleagues uses end-to-end RNA sequencing by Nanopore to examine the effect of oxidative stress on RNA length. They observe transcript shortening under arsenite stress that could be rescued through XRN1 or G3BP1/2 knockdown. The data presented in this paper has interesting implications for the field of mRNA decay, but the authors need to clarify their methodology and interpretations at times. Some experimental issues impact the robustness of their conclusions.

    Major comments:

    (1)    As an overall comment of the manuscript, the way in which the data is presented can sometimes be confusing to the reader. For example, I found the bar graphs in 2b and 3e to be unclear. One way to make the data clearer would be to pull a couple of examples of unchanged transcripts and significantly changed transcripts (as in supplemental figure 2) and use those in a paired dot plot or scatter plot. Those same genes can also be highlighted in all the plots so the reader can easily understand how different plots relate to each other.

    (2)    The authors can end-to-end sequence transcripts by ligating an adapter to 5' monophosphates. As there was no mention of an uncapping step in the protocol, this indicates that the end-to-end sequencing occurs only in transcripts that are already uncapped and in the process of decay. Thus, as written, the authors are only comparing the lengths of RNA decay intermediates during their analyses. That detail is not made clear in the text, but it's an important fact to consider when interpreting the results. Furthermore, it raises some questions about the quality and curation of the library:  What's the efficiency of the ligation step? What percentage of the entire library becomes ligated? Does the population of adaptor-ligated transcripts increase in the XRN1 knockdown due to an accumulation of uncapped transcripts? What is the expected rate of aborted sequencing in Nanopore? Is it possible to identify what percentage of the library is capped mRNAs? Also, what percent of the library is sequenced? Is there a possible bias towards shorter transcripts being sequenced? There is still a lot of quality information to be obtained with the adaptor ligated library, but it's important to contextualize it properly.

    (3)    The authors do not observe polyA tail shortening prior to 5' decay. However, the protocol includes a polyA enrichment step. This step would bias the library to the presence of tails and possibly to longer tails. Given this bias, I'm not sure it's possible to draw robust conclusions about the length of polyA tails without a CNOT knockdown.

    (4)    This manuscript has an interesting observation of G3BP1/2 knockdown rescues transcript length. The conclusion that the effect is "dependent on stress granule formation" is a bit premature. To differentiate a G3BP1/2 specific result from a stress granule specific result I recommend an experiment in which stress granules can form in the absence of G3BP, such as with osmotic stress through sorbitol or using a synthetic condenser that is not G3BP (see Yang et al. 2020, Taylor Lab)

    Minor comments:

    (1)    According to figure 3e, XRN1 knockdown under arsenite stress results in lengthened transcripts. A rescue of the length in knockdown would be logical, but the data as it is presented indicate a dramatic increase in length. I'd like the authors to discuss possible mechanisms to explain this extremely interesting result. In addition, the effects of XRN1 knockdown in an unstressed condition would be important for comparison.

    (2)    How is "differential transcript length" defined?

    (3)    During stress, only an approximate 10% of bulk mRNA molecules accumulate in stress granules and only 185 genes have more than half their molecules localizing to stress granules. I think this manuscript would be enriched by a discussion on if those results reflect those numbers and if not, through which mechanisms stress granules would have such bulk effects while only directly localizing a small portion of transcripts.

    Competing interests

    The authors declare that they have no competing interests.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10999027.

    In this manuscript, the authors employed an end-to-end direct RNA sequencing technique called TERA-seq to study RNA decay at the single nucleotide resolution during cellular stress. Their sequencing analyses showed that a subset of RNAs have shortened 5' ends upon cellular stress conditions that is independent of their 3' end de-adenylation. They suggested that this 5' end decay is dependent on XRN1 and G3BP1/2 by showing that 5' end shortening can be rescued after XRN1 knockdown and ΔΔG3BP1/2, respectively. By comparing with publicly available datasets describing the stress granule transcriptome, they further proposed that stress granule formation is required for 5' end decay since these shortened RNAs mostly reside in stress granules and absence of stress granules in ΔΔG3BP1/2 cells did not lead to rapid decay of these mRNAs.

    Overall, findings from this manuscript are of interest to the field but with the current data in the manuscript, it is difficult to interpret the robustness of the conclusions made by the authors. Some conclusions might be an over-interpretation of the data and would require further experimental validation.

    Major points:

    1.     A brief look at the genes in Sup. Table 4 suggested that majority of the genes have a length of ~2 kb. For known long RNAs such as MALAT1 (8 kb) and NORAD (5.3 kb), their mean length is also around 1-2 kb, even in the unstressed condition. This is also evident in Supple. Fig. 2a & 2b where the significantly shortened transcripts are longer than non-significantly shortened transcripts. This suggests that either the sequencing technique used here might be biased for a certain transcript length or the transcripts being sequenced are mostly decay intermediates. The authors should clarify this discrepancy to strengthen their conclusions. If the authors are solely sequencing RNA decay intermediates, the work is still of interest but this needs to be made clear and the interpretations clarified.

    2.     The authors did not use any orthogonal methods to validate their sequencing results. They could validate some of their top candidates by performing smFISH on both the 5' end and coding region of the transcript in unstressed and NaAs treatment. Alternatively, they could perform Northern blots to show that transcripts are shortened, and this is specifically due to 5' end decay.

    3.     It is not appropriate to conclude that their data showing an association between G3BP1/2 and RNA decay demonstrates that 5' end RNA decay is dependent on stress granules (also indicated by the title of the manuscript). Although ΔΔG3BP1/2 cells are deficient in stress granule formation, G3BP1/2 and stress granule function could be independent of each other since stress granule formation can be rescued in ΔΔG3BP1/2 cells, and G3BP proteins can have additional functions. To delineate between G3BP1/2 and stress granule dependence, the authors could test 5' end shortening in ΔΔG3BP1/2 cells treated with sorbitol which forms stress granules independent of G3BP1/2. Alternatively, they could transfect ΔΔG3BP1/2 cells with FKBPF36M,2ZnF-G3BP1 constructs (Yang et al., 2020, Cell) that also produces stress granules independent of G3BP1/2 function.

    4.     It is difficult to interpret Figure 3e. From Figure 3b & 3c, it suggests that siXRN1 rescued 5' end shortening during stress compared to unstressed. However, Figure 3e shows that for the significantly shortened genes, siXRN1 not only rescued 5' end shortening but led to even longer transcripts. If this is the conclusion, the authors should address this observation in the manuscript. Otherwise, it would be better to change the data presentation in Figure 3e to make a clearer and more straightforward point.  

    Minor points:

    1.     Missing "." in figure citation for line "stress-induced significantly shortened RNAs (Fig 3e) …"

    2.     Wrong citation of sup. fig. in line "Similarly, no difference was observed upon XRN1 silencing (Sup. Fig. 1a) …"

    3.     Difficult to see the dashed lines under the solid lines in Fig 4a. Could consider making the solid lines a lighter shade to show that both lines overlapping.  

    Competing interests

    The authors declare that they have no competing interests.

  3. Version published to 10.7554/elife.96284.1 on eLife
  4. Version published to 10.7554/elife.96284 on eLife
  5. eLife

    eLife assessment

    This valuable study describes mRNA shortening during cellular stress and interestingly observes that this shortening is dependent on localization in stress granules. Surprisingly, this mRNA shortening does not appear to require the shortening of polyA tails. These are in principle novel findings, but the evidence for them is currently incomplete. Additional experiments would help bolster confidence in how the authors interpret their data.

  6. eLife

    Reviewer #1 (Public Review):

    Summary:

    In this manuscript, the authors employed direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. They conclude that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Contrary to the literature, they found that, unlike typical RNA decay models in normal conditions, stress-induced RNA decay is dependent on XRN1 but does not depend on the removal of the poly(A) tail. The findings presented are interesting but a substantial amount of work is needed to fully establish these paradigm-shifting findings.

    Strengths:

    These are paradigm-shifting observations using cutting-edge technologies.

    Weaknesses:

    The conclusions do not appear to be fully supported by the data presented.

  7. eLife

    Reviewer #2 (Public Review):

    In the manuscript "Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress", Dar, Malla, and colleagues use direct RNA sequencing on nanopores to characterize the transcriptome after arsenite and oxidative stress. They observe a population of transcripts that are shortened during stress. The authors hypothesize that this shortening is mediated by the 5'-3' exonuclease XRN1, as XRN1 knockdown results in longer transcripts. Interestingly, the authors do not observe a polyA-tail shortening, which is typically thought to precede decapping and XRN1-mediated transcript decay. Finally, the authors use G3BP1 knockout cells to demonstrate that stress granule formation is required for the observed transcript shortening.

    The manuscript contains intriguing findings of interest to the mRNA decay community. That said, it appears that the authors at times overinterpret the data they get from a handful of direct RNA sequencing experiments. To bolster some of the statements additional experiments might be desirable.

    A selection of comments:

    (1) Considering that the authors compare the effects of stress, stress granule formation, and XRN1 loss on transcriptome profiles, it would be desirable to use a single-cell system (and validated in a few more). Most of the direct RNAseq is performed in HeLa cells, but the experiments showing that stress granule formation is required come from U2OS cells, while short RNAseq data showing loss of coverage on mRNA 5'ends is reanalyzed from HEK293 cells. It may be plausible that the same pathways operate in all those cells, but it is not rigorously demonstrated.

    (2) An interesting finding of the manuscript is that polyA tail shortening is not observed prior to transcript shortening. The authors would need to demonstrate that their approach is capable of detecting shortened polyA tails. Using polyA purified RNA to look at the status of polyA tail length may not be ideal (as avidity to oligodT beads may increase with polyA tail length and therefore the authors bias themselves to longer tails anyway). At the very least, the use of positive controls would be desirable; e.g. knockdown of CCR4/NOT.

    (3) The authors use a strategy of ligating an adapter to 5' phosphorylated RNA (presumably the breakdown fragments) to be able to distinguish true mRNA fragments from artifacts of abortive nanopore sequencing. This is a fantastic approach to curating a clean dataset. Unfortunately, the authors don't appear to go through with discarding fragments that are not adapter-ligated (presumably to increase the depth of analysis; they do offer Figure 1e that shows similar changes in transcript length for fragments with adapter, compared to Figure 1d). It would be good to know how many reads in total had the adapter. Furthermore, it would be good to know what percentage of reads without adapters are products of abortive sequencing. What percentage of reads had 5'OH ends (could be answered by ligating a different adapter to kinase-treated transcripts). More read curation would also be desirable when building the metagene analysis - why do the authors include every 3'end of sequenced reads (their RNA purification scheme requires a polyA tail, so non-polyadenylated fragments are recovered in a non-quantitative manner and should be discarded).

    (4) The authors should come to a clear conclusion about what "transcript shortening" means. Is it exonucleolytic shortening from the 5'end? They cannot say much about the 3'ends anyway (see above). Or are we talking about endonucleolytic cuts leaving 5'P that then can be attached by XRN1 (again, what is the ratio of 5'P and 5'OH fragments; also, what is the ratio of shortened to full-length RNA)?

    (5) The authors should clearly explain how they think the transcript shortening comes about. They claim it does not need polyA shortening, but then do not explain where the XRN1 substrate comes from. Does their effect require decapping? Or endonucleolytic attacks?

    (6) XRN1 KD results in lengthened transcripts. That is not surprising as XRN1 is an exonuclease - and XRN1 does not merely rescue arsenite stress-mediated transcript shortening, but results in a dramatic transcript lengthening.

  8. eLife

    Reviewer #3 (Public Review):

    The work by Dar et al. examines RNA metabolism under cellular stress, focusing on stress-granule-dependent RNA decay. It employs direct RNA sequencing with a Nanopore-based method, revealing that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy but is independent of the shortening of the poly(A) tail. This decay, however, is dependent on XRN1 and enriched in the stress granule transcriptome. Notably, inhibiting stress granule formation in G3BP1/2-null cells restores the RNA length to the same level as wild-type. It suppresses stress-induced decay, identifying RNA decay as a critical determinant of RNA metabolism during cellular stress and highlighting its dependence on stress-granule formation.

    This is an exciting and novel discovery. I am not an expert in sequencing technologies or sequencing data analysis, so I will limit my comments purely to biology and not technical points. The PI is a leader in applying innovative sequencing methods to studying mRNA decay.

    One aspect that appeared overlooked is that poly(A) tail shortening per se does lead to decapping. It is shortening below a certain threshold of 8-10 As that triggers decapping. Therefore, I found the conclusion that poly(A) tail shortening is not required for stress-induced decay to be somewhat premature. For a robust test of this hypothesis, the authors should consider performing their analysis in conditions where CNOT7/8 is knocked down with siRNA.

    Similarly, as XRN1 requires decapping to take place, it necessitates the experiment where a dominant-negative DCP2 mutant is over-expressed.

    Are G3BP1/2 stress granules required for stress-induced decay or simply sites for storage? This part seems unclear. A very worthwhile test here would be to assess in XRN1-null background.

    Finally, the authors speculate that the mechanism of stress-induced decay may have evolved to relieve translational load during stress. But why degrade the 5' end when removing the cap may be sufficient? This returns to the question of assessing the role of decapping in this mechanism.

  9. Version published to 10.1101/2023.08.31.555629v2 on bioRxiv
  10. Version published to 10.1101/2023.08.31.555629v1 on bioRxiv