Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia
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Evaluation Summary:
This study reports a novel link by which specific cellular mRNAs, that contain internal ribosome sites (IRES), are made competent for translation in paraspeckles in the nucleus. The data showed that a long noncoding RNA, Neat1, is the major player to add transacting factors to the internal ribosome entry site located in fibroblast growth factor 1 mRNAs in the nucleus. This event occurs during hypoxia in mouse cardiomyocytes and is, thus, relevant to gene expression during angiogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here, we searched for IRES trans -acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1 , an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54 nrb and PSPC1 as well as nucleolin and RPS2, two p54 nrb -interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response.
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Reviewer #2 (Public Review):
Godet et. al have attempted to identify the cellular components of what is known as an IRESome, and conclude that paraspeckles are the sites of IRES action. They found that IRES accessory proteins known as ITAFs concentrate during hypoxia at that paraspeckle sites and are important for IRES-mediated translation. The authors show that the long non-coding RNA Neat1, and in particular isoform 2 of NEAT1, is a universal essential component that can recognize almost all cellular IRESs and contributes to their translation during the stress response in angiogenesis and/or cardio-protection. In summary, the authors propose a novel and very interesting concept, but one which is still incomplete and will require additional experimentation in order to convincingly conclude that the lncRNA NEAT1 is required for IRES …
Reviewer #2 (Public Review):
Godet et. al have attempted to identify the cellular components of what is known as an IRESome, and conclude that paraspeckles are the sites of IRES action. They found that IRES accessory proteins known as ITAFs concentrate during hypoxia at that paraspeckle sites and are important for IRES-mediated translation. The authors show that the long non-coding RNA Neat1, and in particular isoform 2 of NEAT1, is a universal essential component that can recognize almost all cellular IRESs and contributes to their translation during the stress response in angiogenesis and/or cardio-protection. In summary, the authors propose a novel and very interesting concept, but one which is still incomplete and will require additional experimentation in order to convincingly conclude that the lncRNA NEAT1 is required for IRES mediated mRNA translation activity.
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Evaluation Summary:
This study reports a novel link by which specific cellular mRNAs, that contain internal ribosome sites (IRES), are made competent for translation in paraspeckles in the nucleus. The data showed that a long noncoding RNA, Neat1, is the major player to add transacting factors to the internal ribosome entry site located in fibroblast growth factor 1 mRNAs in the nucleus. This event occurs during hypoxia in mouse cardiomyocytes and is, thus, relevant to gene expression during angiogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript investigates a topic of general interest to translational regulation - IRES function during hypoxia. The authors propose that nuclear paraspeckles serve as areas where cellular IRESes acquire their ITAFs and that this subsequently enables them (the IRESes) to be appropriately expressed. Among the components of the paraspeckles that the authors find associated with the FGF1 IRES is the lncRNA, Neat1, and a few resident proteins. The strengths of the current study is that the presented experiments are generally well presented and described. The manuscript is well written. The experiments cover a wide breadth in the area of FGF1 IRES activity/regulation. The weaknesses lies in several instances where correlation between datasets are taken to imply direct cause-effect relationships. Some …
Reviewer #1 (Public Review):
The manuscript investigates a topic of general interest to translational regulation - IRES function during hypoxia. The authors propose that nuclear paraspeckles serve as areas where cellular IRESes acquire their ITAFs and that this subsequently enables them (the IRESes) to be appropriately expressed. Among the components of the paraspeckles that the authors find associated with the FGF1 IRES is the lncRNA, Neat1, and a few resident proteins. The strengths of the current study is that the presented experiments are generally well presented and described. The manuscript is well written. The experiments cover a wide breadth in the area of FGF1 IRES activity/regulation. The weaknesses lies in several instances where correlation between datasets are taken to imply direct cause-effect relationships. Some experiments take several days to set-up (eg, knock-downs) and it thus becomes difficult to establish such direct cause-effect relationships versus effects due to secondary causes.
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Reviewer #3 (Public Review):
This study searched for IRES transacting-factors (ITAFs) that control the translation of the IRES in fibroblast growth factor FGF1 mRNA during normoxia and hypoxia in mouse cardiomyocytes. Because it has been known that several ITAFs locate to nuclear paraspeckles, the authors examined roles for a long noncoding RNA, NEAT1, that is located to these speckles, in the activation of the FGF1-IRES. Using depletion studies it was shown that NEAT1 indeed modulation of FGF1 IRES activity. Using a tagged version of p54nrb, which interacts with NEAT1, several interacting proteins were discovered by mass spectrometry. SiRNA-mediated depletion of the mRNAs encoding some of these proteins (i.e. RPS2, hnRNPM, nucleolin) showed a very modest decrease in IRES activity during normoxia, but less so during hypoxia. Finally, …
Reviewer #3 (Public Review):
This study searched for IRES transacting-factors (ITAFs) that control the translation of the IRES in fibroblast growth factor FGF1 mRNA during normoxia and hypoxia in mouse cardiomyocytes. Because it has been known that several ITAFs locate to nuclear paraspeckles, the authors examined roles for a long noncoding RNA, NEAT1, that is located to these speckles, in the activation of the FGF1-IRES. Using depletion studies it was shown that NEAT1 indeed modulation of FGF1 IRES activity. Using a tagged version of p54nrb, which interacts with NEAT1, several interacting proteins were discovered by mass spectrometry. SiRNA-mediated depletion of the mRNAs encoding some of these proteins (i.e. RPS2, hnRNPM, nucleolin) showed a very modest decrease in IRES activity during normoxia, but less so during hypoxia. Finally, the authors showed that effects of NEAT1 on translation were specific for IRES-containing mRNAs that function during angiogenesis and cardioprotection. While effects of NEAT1 on FGF1 translation is supported by solid data, roles for NEAT1-interacting ITAFs is less clear. However, pre-assembly of translation-competent FGF1 in nuclear paraspeckles is a novel finding that may be very relevant in cardiomyocytes.
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