Viral manipulation of mechanoresponsive signaling disassembles processing bodies

  1. Elizabeth L. Castle
  2. Carolyn-Ann Robinson
  3. Pauline Douglas
  4. Kristina D. Rinker
  5. Jennifer A. Corcoran

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Abstract

Processing bodies (PBs) are ribonucleoprotein granules that suppress cytokine mRNA translation that are targeted for disassembly by many viruses. Kaposi’s sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposi’s sarcoma, and a PB-regulating virus. The virus encodes Kaposin B (KapB), which induces actin stress fibres (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB and mechanoresponsive PB disassembly also required YAP. Using the viral protein KapB, we identified a new consequence of the exposure of cells to mechanical forces that alter actin dynamics and activate YAP, namely the disassembly of PBs.

Importance

For the first time, we demonstrate that processing bodies (PBs), cytoplasmic sites of RNA decay, are regulated by mechanical signaling events that alter actin dynamics and that this requires the mechanoresponsive transcription factor, YAP. Using the overexpression of a viral protein called KapB, known previously to mediate PB disassembly, we show that actin stress fibers (SFs) and the mechanoresponsive transcription factor, YAP, are required for PB loss. We also show that other established mechanical signals (shear stress or stiff extracellular matrix) that lead to the formation of SFs and activate YAP also cause PB disassembly. This is important because it means that KapB activates, from the inside out, a pathway that links cell shape to post-transcriptional gene regulation via cytoplasmic PBs.

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  1. eLife

    ###Reviewer #3:

    This report examines the mechanisms by which the KSHV KaposinB (KapB) protein causes disassembly of processing bodies (PBs) in HUVECs. Convincing data is presented showing that mDia1 and ROCK, factors downstream of RhoA, are necessary for PB disassembly in HUVEC cells. Data suggesting cofilin enhances KapB PB disassembly is less convincing. Over-expression of actinin-1 or directly activating actomyosin contraction favored PB disassembly, implicating mechano-responsive signaling components. Analysis of YAP, a mechano-responsive transcription factor showed that levels were elevated in cells expressing KapB and its knockdown rescued PB formation in KapB expressing cells. Expression of constitutively active YAP promoted PB disassembly, similar to KapB, although it did not reproduce the stabilization of ARE-containing mRNAs seen in KapB-expressing cells. Interestingly, subjecting cells to shear stress or increasing the stiffness of the matrix on which they grow, both thought to activate YAP, recapitulated the PB disassembly phenotype seen in cells expressing KapB and knockdown of YAP abolished this.

    These are interesting and exciting results that further illuminate the mechanisms by which a viral protein perturbs PB function. Perhaps even more exciting is the finding that mechano-sensitive signaling pathways can influence PB formation (and perhaps) function. The data are of high quality and support the major conclusions of the study. However, a couple items could be addressed that raised questions with me. First, there is some question as to whether or not the impact shown is a general effect on PBs as a whole or just on the HEDLS marker that is used exclusively in the study. Showing that another PB marker (or two) behaves similarly would support this conclusion. Perhaps doing this for a few key conditions- such as the shear-stress and expression of constitutively active YAP would be possible. The authors conclude, based on a TEAD-Luc reporter assay, that YAP transcriptional activity is not induced even though it appears to be up significantly compared to controls (Fig S5A, left panel). Could they elaborate on how they arrived at this conclusion? The argument that levels of phospho-YAP are not increased in KapB-expressing cells is not supported by the data. While the ratio may not be different, the total amount of phospho-YAP is clearly elevated, as are total YAP levels. Throughout the manuscript, can the authors comment on the impact of knockdowns on cell viability, morphology, if any?

  2. eLife

    ###Reviewer #2:

    The authors demonstrate that disappearance of P-bodies from cells expressing a KSHV protein, KapB, requires factors regulating actin contractility, mechanosensation and YAP - but does not require the transcriptional regulatory activity of YAP. The function of P-bodies has long been contentious, and the endogenous mechanisms regulating P-body assembly vs. disassembly are still being elucidated. Many studies of P-body dynamics have relied on treatment with sodium arsenite, global translational inhibition, etc. This study therefore has the potential to add significantly to our understanding of P-body disassembly mechanisms and improve our understanding of the role of these ribonucleoprotein granules in cells. Several points of data presentation and interpretation may benefit from clarification.

    1. The introduction and discussion present P-bodies as sites of decay of ARE-containing mRNAs, a long-accepted model of P-body function. However, building on well-established observations from the Izaurralde lab that RNA decay is uncoupled from P-body formation, recent work by Parker, Singer, and Chao utilizing single-molecule imaging of 5' end decay provided clear support for cytosolic localization of RNA decay events, with no decay occurring inside P-bodies, strongly supporting a storage/translational repression role for P-bodies rather than a role in decay. The authors then attempt to provide a complex explanation of the observation that constitutively active YAP decouples P-body disassembly from ARE mRNA stability, rather than considering this result in the context of alternative P-body models.

    2. It is unclear why, in Fig. 1B (middle panel), there is a large, statistically significant increase in P-bodies per cell in vector-expressing cells - which do not express KapB - treated with shDia1-1 over shNT - but not with shDia1-2. Is this due to the more efficient silencing of mDia1 expression by shDia1-1, and does mDia1 have a KapB-independent effect on P-bodies? Or does this suggest off-target shRNA effects?

    3. It appears throughout the manuscript that there is always far more dispersion in P-body numbers in experimental (either shRNA or inhibitor-treated) cells than in control cells, though this may be an artefact of the fold-change calculation in which the authors normalize control cells to 1.0 and present no estimate of variance. Especially for experiments in which p values are close to the cutoff for significance, meaningful analysis of variance in all measurements is important and presentation of the raw data pre-normalization may be helpful.

    4. In Figure 4A, are the KapB expressing cells larger than the vector-expressing cells, or is a higher magnification used? The nuclei appear nearly double in diameter. In the immunofluorescence experiment, no other control marker is imaged to support the assertion that YAP signal is selectively increased by KapB expression. No image quantitation is performed to support the assertion that "nuclear:cytoplasmic YAP was not markedly increased". Quantitation across multiple fields of view (and discussion of how many cells were utilized in the image analysis) rather than presentation of a single image would address these concerns. The authors' observation that the fraction of phosphorylated YAP, as measured by Western blotting in Fig. 4B, decreases in KapB expressing cells appears incongruent with the stated lack of change in cytoplasmic:nuclear YAP in KapB vs. vector expressing cells (Fig. 4A).

    5. While I appreciate that the authors have utilized the luciferase assay in multiple studies, direct measurement of the luciferase reporter mRNA stabilities should be performed to differentiate between changes in stability of the ARE mRNA vs. selective translational repression of the ARE mRNA in this specific experimental context.

    6. "Comparison of the transcriptomic data from HUVECs subjected to shear stress from Vozzi et al (2018) (Accession: GEO, GSE45225) to entries in the ARE-mRNA database (Bakheet, Hitti, and Khabar 2017) showed a 20% enrichment in the proportion of genes that contained AREs in those transcripts that were upregulated by shear stress." This comparison (1) lacks any measure that this enrichment is significant, and (2) relies on a single steady-state microarray measurement, and therefore does not accurately report on RNA decay rates/permit conclusions about RNA dynamics.

    7. It is impossible for the reviewer to assess "unpublished data" on autophagy cited in the discussion.

  3. eLife

    ###Reviewer #1:

    In this manuscript the authors show that the oncogenic transcription factor YAP is an important factor in the signaling pathway from the Kaposi Sarcoma virus protein KapB via the host cell GTPase RhoA down to the disassembly of processing bodies (PBs). This is in principle an interesting finding. However, the connection between KapB and PB-disruption, between YAP and the Rho pathway, Kaposi KapB and the Rho pathway, as well as the connection between Kaposi virus infection and YAP (and Rho) have been described before. Therefore, this connection alone does not come as a surprise. New mechanistic insight into how exactly YAP contributes in PB disruption is unfortunately missing.

    1. A bit contradictory is that the last author in 2015 was first authoring a paper in which they did not receive a significant PB-rescue with ROCK inhibitor, leading them to the conclusion that contractility and PB-disruption are independent events downstream of RhoA activity. In the current manuscript they now revise this and convince the reader that PB disruption involves contractility (which is also more in line with earlier work (Takahashi et al., 2011)).

    2. The fact that contractility leads to YAP activation is known, but the authors now convincingly show that this does not happen in parallel, but that PB disruption depends on YAP activation. Therefore, the most interesting aspect is that RNAi-mediated removal of YAP leads to suppression of P-body disruption. This finding places YAP as an essential intermediate between contractility and PB-disruption. This reviewer really likes this finding but requests that the authors follow this path a little further and add to the mechanism.

    i) Is it based on a protein-DNA interaction of YAP, i.e. does YAP need to act as transcription factor to induce PB dissolution? And what transcripts would then be induced and be required for PB disruption or dispersal? Could it be something like DICER RISC (Chaulk et al., 2014)? The authors delineate that this first option is less likely to them but no experimental proof is provided.

    ii) The effect of YAP on PBs might be based on a protein-RNA interaction or

    iii) It might depend on a protein-protein interaction between YAP and an unidentified partner?

    iv) Finally, one could ask if PB dispersal is connected to an induction of autophagy?

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This report examines the mechanism by which the KSHV KaposinB (KapB) protein causes disassembly of processing bodies (PBs) in HUVECs. The authors show that the oncogenic transcription factor YAP is an important component in the signaling pathway of KapB of the oncogenic herpesvirus Kaposi's Sarcoma herpesvirus, which involves the host cell GTPase RhoA, leading to disassembly of processing bodies (PBs).

  5. Version published to 10.1101/2020.05.15.091876 on bioRxiv