C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in  Functional and structural distinct axonal and dendritic compartments demand specific regulation of mitochondrial distribution. While most of previous studies examined mitochondrial transport in cultured neurons or at a certain segment of axon, we genetically ablated five motors and four regulators and study their roles in a sensory neuron that has two microtubule polarity distinct dendrites and an axon. We showed that dynein regulates mitochondrial distribution in neurites based on microtubule polarity regardless of axon or dendrite, while KIF5, but not the other kinesin motors, mediates an axon-specific mitochondrial transport. We surprisingly found that CRMP/UNC-33 is critical for axonal mitochondria and it supports a stable microtubule population to form a spindle-like structure in neuronal cell body as tracks for trafficking. These results indicate that CRMP/UNC-33 promotes high-order organization of microtubule bundles in neurons, which are cells without a clear centralized microtubule organization center but still possess highly organized microtubule bundles. As defects of mitochondrial trafficking are implicated in most of the neurodegeneration diseases, this study manifests specific roles of motors and regulators in distinct axonal and dendritic compartments, and it further reveals how the microtubule network forms in the neuronal cell body. Neurons are polarized cells with functionally distinct cellular compartments including the somatodendritic and axonal compartments. In the somatodendritic compartment, branched dendrites cover receptor fields for receiving signals, while typically a long and sheer axon reaches target region in order to output signals. Apart from most other cells, the cellular processes of neurons are particularly long. For example, the axonal length of some motor neurons can be more than one meter long in mammals . Hence, in vitro or in cultured cells X, ntuIs2III, ntuIs6III, ntuIs10. Extrachromosomal lines used were: ntuEx22, 23, ntuEx24-26, ntuEx27-30, 126, ntuEx35, ntuEx178, ntuEx179, ntuEx182, ntuEx185, wyEx5216 [Podr-1::GFP and Pmyo-2::GFP were used as co-injection markers.Integrated transgenes used were: UNC-104E2K::GFP 10-1::RFP] . Podr-1:S. McCarroll and C. I. Bargmann, personal communication). The Pdes-2 promoter was used for PVD-specific expression, and Pmec-4 promoter was used for touch receptor neuron expression. Pdes-2 promoter fragment was amplified as previously described We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Andrew D. ChisholmAssociate EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The manuscript by Chan-Yen Ou group entitled “CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon” evaluated how mictochondrial distribution in different compartments of neurons is regulated. They examined mitochondrial motors and transport regulators for their roles in mitochondrial transport using the PVD neuron as a model. This is an important topic, given that mitochondrial trafficking is essential for neuronal function and its dysregulation is involved in various neurodegenerative diseases.Despite the characterization of factors regulating mitochondrial transport, how is this regulated in different cell compartments is less clear. The authors established a transgenic reporter for mitochondrial distribution in PVD neurons and examined how mitochondrial distribution was affected by loss of function of kinesins , dynein (dhc-1), and transport regulators . They revealed distinctive roles of these genes in mitochondrial transport and analyzed how they genetically interact. As unc-33 is known to regulate microtubule organization, they went ahead to test whether unc-33 impact motor polarized mitochondrial distribution by regulating general cytoskeletal arrangement. They concluded that UNC-33 selectively stabilizes axonal microtubules to promote UNC-116-mediated mitochondrial transport to axons. Overall, I found the genetics experiments to be quite compelling. However, I believe a few issues will need to be addressed.Major concerns:1. The authors established a very nice genetic reporter to study mitochondrial transport in PVD neurons and identified specific roles of the motors and regulators in different compartments of the neuron. Are these findings neuron type-specific, or can these findings be applied to other neuron types, such as touch sensory neurons and DA9 neuron, which have long been used to study vesicular trafficking and microtubule organization?2. The authors have concluded that “UNC-33 sticks MT bundles together to form a spindle-like high-order structure in the cell body and selectively maintains stabilized MTs for KIF5”. They did not provide images showing UNC-33 and UNC-116-rigor-GFP, but based on the Mossci reporter of UNC-33::GFP, the subcellular localization pattern of UNC-33 does not match with UNC-116-rigor-GFP pattern (i.e. spindle-like pattern in soma).3. They propose that UNC-33 adheres stable MT bundles to some structure of cell body cortex, which is likely to be the F-actin network beneath the cell membrane. Can they test this?4. They have shown that UNC-33 is localized in soma, axon and dendrites, then why UNC-33 selectively stabilize microtubules in axons? They discussed that this might not be due to microtubule polarity. Testing this in a different neuron type might help too.5. Can microtubule targeting agents rescue the defects in unc-33 mutant?Minor issue:The figures provided for review are in extremely low resolution, making it super hard to read and evaluate the data.Typo in line 342-343: “Whether dose CRMP/UNC-33 regulate mitochondrial transport by directly promoting kinesin-1 activity?”Reviewer #2: The manuscript by Ying-Chun Chen et al addressed how mitochondria is transported differentially in axon and dendrites using PVD neuron model in worm. They used mitochondrial reporter TOMM20::GFP and mutants deleting microtubule-based motors and their regulatory subunits to address the above-mentioned question. While it is really appreciated that PVD neuron would be a good system to look at the mitochondrial transport in neuron and authors develop a nice system to study this area. Using this system, the authors showed that KIF5/UNC-116 and dynein/DHC-1 is important for transport of mitochondria in axon and dendrites respectively. The mitochondrial adaptor Miro-1 plays an important role in mitochondrial distribution in all the neurites. According to the authors, more intriguing is the finding that CRMP family protein UNC-33 plays an important role in this process by regulating the organization of microtubule cytoskeleton in axon and dendrites. I completely agree that it is a nice characterization of this new system for studying mitochondrial transport. However, a major concern is that lack of new insight this study gives on mitochondrial distribution in neuronal cell.1) For example, the role of KIF5/UNC-116 , Miro-1  and dynein  is well established in axonal and dendritic transport of mitochondria. There are many other reports on the role of kinesin-1 and dynein in axonal transport of mitochondria.2) The major finding that UNC-33 is important for mitochondrial transport lacks sufficient insight. For example, ‘Maniar et al 2011 Nat Neurosc’ clearly demonstrated that UNC-33 is important for establishment of microtubule polarity in axon and dendrites of PVD neuron. In the absence of the same MTs are disorganized and affects distribution of motors and cargoes in synapse and dendrites. Apart from that also other studies that highlight the importance of UNC-33/CRMP in neuronal microtubule organization in C. elegans  and vertebrate model. Essentially, it’s important that authors highlight their findings that advances this field. On that basis, I feel that this study would be more suitable for elsewhere.3) The authors may highlight how UNC-33 might be specifically important for mitochondrial transport? Can they provide some evidence by disturbing the specific function of UNC-33 related to mitochondria that only mitochondrial transport is affected without altering the distribution of other components such as RAB-3.4) The UNC-33L isoform was shown to regulate microtubule organization earlier . Although authors suggest M and S isoform does not rescue, no quantitative data is presented.5) In figure-5, the authors claim that UNC-33 is involved in organization of UNC-116-bound microtubule tracks. How can they be sure that the other microtubule population is unaffected?6) I suggest the authors that they proofread the manuscript for the language and grammatical errors. At times, it’s difficult to follow. It needs a major rewriting.Reviewer #3: This work describes the roles of dynein and kinesin MT motor proteins, adapters, and the MT organizing molecule UNC-33/CRMP in mitochondrial distribution in neurons. The PVD sensory neuron has process with MTs of distinct polarities: + end out axon and posterior dendrite; and - end out anterior dendrite. They find that specific motor proteins and adapters are responsible for mitochondrial distribution in these processes. They also find that UNC-33/CRMP is required to establish a unique MT structure in the cell body utilized by UNC-116/KIF5 for mitochondrial entry into processes from the cell body. The mitochondrial distribution assay in the PVD neuron is nice, and is the basis of many experiments in the manuscript. The live imaging of mitochondrial entry into processes was good. These are interesting and significant results. The experiments are in general well designed and controlled, with appropriate statistical analyses. The figures, however, were of too low a resolution to assess some of the claims made by the authors in the text. My comments to improve the manuscript are below.1) The resolution of the figures in the pdf copy that I downloaded was really low, making it difficult in some instances to see in micrographs what the authors describe in the text. Likewise, some of the graphs and data figures were small and at this resolution, difficult to see clearly.2) Many of the experiments rely on quantifying mitochondrial prevalence in distinct processes and sub-sections of processes. The authors make claims about increased or decreased mitochondrial movement. However, it is also possible that more or fewer overall mitochondria are being generated. This should be addressed in each experiment.3) Add a reference for the unc-33(mn407) deletion allele or add a figure with the deletion data.4) The argument about unc-16/JIP3 gating rather than minus end specificity would be bolstered by analyzing total mitochondria in this mutant.5) The authors find that mutations in the dhc-3 dynein heavy chain partially suppress defects in unc-33 mutants. Because suppression is not complete, the authors argue that dhc-3 and unc-33 act independently and that unc-33 is unlikely to regulate mitochondrial transport via dhc-3. Is the suppression significant compared to unc-33 alone? If so, I would argue that this suggests that they do interact, and that dhc-3 might be one of multiple unc-33 partners in mitochondrial transport.6) Defects in the unc-33; unc-116 double mutant are increased relative to single mutants. Is this a synergistic effect, or additive? If it is synergistic , this suggests parallel, converging pathways. If it is additive, this suggests mechanistically independent pathways. This should be addressed, as it gets at mechanism.7) The authors mention that MT polarity is disrupted in some mutants. This seems like an important experiment to perform for all mutants, but especially for unc-33. Do unc-33 mutants have MT polarity defects?8) The Results section entitled “UNC-33 organizes microtubule bundles in PVD cell body and stabilizes microtubules in axon” is written in an unclear manner. Part of this is due to referring to MTs using distinct terminology. The authors switch between acetylated versus tyrosinated MTs, stable versus dynamic MTs, and those labeled by the unc-116rigor and unc104rigor. I would suggest described the reagents and what they selectively label, and then describe experiments by using the reagent name. For example, instead of“In the absence of UNC-33, the spindle-like acetylated MT bundles were severely diminished in the cell body, and the general level of tyrosinated MTs in the cortical region of the cell body was also decreased, but the circular mesh of tyrosinated MTs was still present around the nucleus .”Use this terminology:“In the absence of UNC-33, the spindle-like UNC-116rigor signal was severely diminished in the cell body, and the general level of UNC-104rigor signal in the cortical region of the cell body was also decreased, but the circular mesh of unc-104rigor was still present around the nucleus .”9) A reference for the rigor technique is missing.10) Given the resolution of the figures, it was impossible for me to see the spindle-shaped UNC-116rigor structure or the circular mesh of unc-104rigor in Figure 5. Because of this, it was impossible for me to assess the effect of unc-33 on these structures.11) Work from my lab has shown that unc-33 influences MT + end distribution in VD growth cones in vivo . The results presented here are consistent with this result. I would ask the authors to consider referencing this work in discussions about known roles of unc-33/CRMP.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article , and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.In the meantime, please log into Editorial Manager at If you have a press-related query, or would like to know about one way to make your underlying data available , please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!Yours sincerely,Andrew D. ChisholmAssociate EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS Geneticswww.plosgenetics.orgTwitter: @PLOSGenetics----------------------------------------------------Comments from the reviewers (if applicable):Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The revised manuscript has fully addressed my comments raised previously. Therefore, I support its publication.Reviewer #2: The authors have appropriately addressed the comments made by the reviewers. Although, I had some reservations on the novelty of this finding as the role of UNC-33 in microtubule organization is well established, I feel that this work very nicely establishes PVD neuron of worm as a model to study mitochondrial transport and see the consequences in various conditions. The authors have done a through characterization of the transport system in this work and therefore it should be published.Reviewer #3: The authors responded appropriately to reveiwer comments. The additional experiments added, including total mitochondrial levels, strengthen the manuscript.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  5 Feb 2021PGENETICS-D-20-01044R1 CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon Dear Dr Ou, We are pleased to inform you that your manuscript entitled "CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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Gon4l/udu gene in different organisms give rise to diverse phenotypes. Although the effects of Gon4l/Udu in transcriptional regulation have been demonstrated, they cannot solely explain the observed characteristics among species. To further understand the function of Gon4l/Udu, we used yeast two-hybrid (Y2H) screening to identify interacting proteins in zebrafish and mouse systems, confirmed the interactions by co-immunoprecipitation assay, and found four novel Gon4l-interacting proteins: BRCA1 associated protein-1 (Bap1), DNA methyltransferase 1 (Dnmt1), Tho complex 1 , and Cryptochrome circadian regulator 3a (Cry3a). Furthermore, all known Gon4l/Udu-interacting proteins—as found in this study, in previous reports, and in online resources—were investigated by Phenotype Enrichment Analysis. The most enriched phenotypes identified include increased embryonic tissue cell apoptosis, embryonic lethality, increased T cell derived lymphoma incidence, decreased cell proliferation, chromosome instability, and abnormal dopamine level, characteristics that largely resemble those observed in reported Gon4l/udu mutant animals. Similar to the expression pattern of udu, those of bap1, dnmt1, thoc1, and cry3a are also found in the brain region and other tissues. Thus, these findings indicate novel mechanisms of Gon4l/Udu in regulating CpG methylation, histone expression/modification, DNA repair/genomic stability, and RNA binding/processing/export.Mutations of the  Gon4l homologous gene in Caenorhabditis elegans, gon-4, is critical for gonadogenesis. In gon-4 mutant nematodes, precursor cells for somatic gonadal tissues are unable to divide or cell division is severely delayed1. After re-expressing exogenous GON-4 protein, fertility can be rescued in gon-4 mutants. In Drosophila, the Gon4l homologous gene, muscle wasted (mute), has been proposed as one of the components of histone locus body. Loss of function in mute gene results in decreased muscle mass and defective terminal differentiation of muscle cells, therefore, suggesting a role for Mute in regulating muscle cell differentiation through controlling histone expression2.Gon4l is a nuclear protein conserved among species. Animal models from invertebrates to vertebrates have shown that the protein Gon4-like (Gon4l) is essential for regulating cell proliferation and differentiation. For example, the Justy mutant mice, which carry a point mutation within the Gon4l gene, exhibit defects in B cell development3. Detailed analysis reveals that a block in the G1/S phase transition results in severely defective differentiation of pre-pro B cells to pro-B cells in these mutant mice4. Moreover, a frameshift mutation in this gene causes proportionate dwarfism in Fleckvieh cattles5, indicating that the Gon4l protein not only affects cell proliferation/differentiation in specific types of tissues but also can impair the growth of an entire organism.For mammals, ugly duckling (udutu24), was first isolated from the Tübingen large-scale screen and exhibits fewer blood cells, a shorter body axis, and defective tail formation6. Another study shows that udusq3 mutants  exhibit arrested primitive hematopoiesis7. The third udu mutant allele, uduvu66, shows an irregular notochord boundary and reduced mediolateral cell polarity at early stages8. Similar to human and mouse GON4L proteins, zebrafish Udu protein is also a nuclear factor. Its N-terminus contains three conserved regions , and its C-terminus consists of two paired PAH (amphipathic α-helix like) repeats and one SANT domain 7. The PAH domain, which has been identified in the Saccharomyces cerevisiae SIN3 gene, has the ability to mediate protein–protein interaction10; and the SANT domain, which is similar to the Myb DNA-binding domain11, may be responsible for regulating chromatin remodeling and accessibility13. Therefore, the protein structure of human/mouse GON4L and zebrafish Udu indicates their roles in regulating gene expression through their incorporation with other proteins.The zebrafish mutant, 14. GON4L in human and Mute in Drosophila are reported to be co-localized with NPAT  in the histone locus body to regulate the expression and processing of cell cycle-regulated histone transcripts16. Transcription levels of histones H3 and H4 are both up-regulated in mute mutant embryos, suggesting that Mute might be responsible for the repression of histone mRNA accumulation2. Additionally, a recent study using a large-scale protein–protein interaction screen in mouse T cells revealed a novel Gon4l-interacting protein, CRAMP1L, in association with the Hippo kinase, MST117. However, whether the interaction among GON4L, CRAMP1L, and MST1 affects gene transcription in regulatory T cells was not further addressed. In contrast to being a transcriptional repressor, GON4L has been shown to interact with YY1 to positively regulate CD24 expression in several human cancer cell lines18. Noteworthy, it is very interesting that Gon4l/Udu may not only exert its function as a transcriptional regulator. Expression of the PAH/SANT domain in synchronized human cells shows that it is co-localized with the BrdU signal in early S phase and with HP1b, a marker for pericentromeric heterochromatin, in late S phase20. These findings suggest a possible function of Gon4l/Udu for participating in DNA replication and also in protecting the epigenetic integrity.Despite the biological importance of Gon4l/Udu, only a few studies have addressed the biochemical interactions between Gon4l/Udu and other proteins, as well as how interactions among these proteins are related to its possible functions. GON4L in mouse B cells can associate with YY1, Sin3a and Hdac1 to form a complex. The interaction among these proteins correlates with the ability of GON4L to repress targeted DNA expression as determined by GAL4-UAS reporter assaybap1, dnmt1, thoc1, and cry3a co-localize with gon4l/udu during development mainly in the brain region and some other specific tissues. Furthermore, using bioinformatic analysis reveals that enriched phenotypes in mutant animals carrying mutations at genes encoding Gon4l/Udu-interacting proteins are similar to those observed in reported Gon4l/udu mutants. These data not only indicate functional cooperation among Gon4l/Udu and its interacting proteins but also provide important links between the molecular functions of Gon4l/Udu and various kinds of reported biological effects.To further understand the possible functions of Gon4l/Udu, we performed yeast two-hybrid (Y2H) screens in this study to discover novel Gon4l/Udu-interacting proteins from prey libraries of both mouse cell lines and zebrafish larvae. We have isolated 56 Gon4l/Udu-interacting candidate proteins and used co-immunoprecipitation to verify 11 of them. We have identified four proteins as novel partners for Gon4l/Udu—Bap1, Dnmt1, Thoc1, and Cry3a—which are functionally related to histone modification, maintenance of DNA methylation after replication, R-looping prevention and RNA processing during transcription, and circadian-regulated DNA repair, respectively. RNA in situ hybridization studies show that 19. In the mouse screen, 29 proteins were identified as interacting with bait 3 , which contains two putative YY1-binding domains, and the C-terminal PAH and SANT domains —were used as bait  and the known functions of Udu, eleven genes from both mouse and zebrafish Y2H data  and Udu with only the PAH/SANT domain (hereafter Udu-P/S) were fused with Flag tag. Since Udu is known as a nuclear factor19, nuclear localization of Udu-full or Udu-P/S was first confirmed using immunofluorescence , BNIP-Sα and BNIP-Sβ, have been reported to be expressed in the cytoplasm and nucleus, respectively27. Interestingly, we found some cells expressed both cytoplasmic and nuclear Bnipl . Therefore, we used BioGRID to find Gon4l-interacting proteins that were identified from large-scale screenings but had never been specifically addressed in the literature. The dataset from mouse studies showed only 3 known Gon4l-interacting proteins, including YY1, Sin3A and Hdac1, all of which can co-regulate transcription of downstream genes  is a database that collects and archives protein, genetic, and physical interactions for model organisms, such as mice and humans  to analyze phenotypes that are enriched by 20 Gon4l-interacting proteins . The proteins used for modPhEA are listed in Supplementary Table https://www.informatics.jax.org), the most enriched phenotypes are related to increased embryonic tissue cell apoptosis (p = 0.028), embryonic lethality (p = 0.014), increased T cell derived lymphoma incidence (p = 0.006), decreased cell proliferation (p = 0.012), chromosome instability (p = 0.018), and abnormal dopamine level (p = 0.014) . Similar to results obtained from mouse phenome, early embryonic lethal was enriched (p = 0.001) in the WBPhenotype database. Other phenotypes—such as nuclear morphology variation early emb (p = 1.2 × 10–5), sister chromatid segregation defective early emb (p = 6.3 × 10–5), embryonic cell morphology variant (p = 3.6 × 10–4), body elongation variant (p = 0.002), programmed cell death variant (p = 0.004), thin (p = 0.004), and reproductive system morphology variant (p = 0.007)—were also enriched by Gon4l-interacting proteins (Supplementary Table Gon4l/udu mutant organisms.We then asked whether the phenotypic changes observed in 4) Table . After f4) Table . However4) Table . Additioudutu24 mutants19, indicating a consequence from the loss of Udu interactions with Bap1, Dnmt1, and/or Thoc1. To further verify this hypothesis, we examined the mRNA expression patterns of bap1, dnmt1, and thoc1 during development. Due to the lack of reported phenotypes about Cry3a mutants in the modPhEA databases, we also checked the expression patterns of cry3a and compared those of four with that of udu at the same stages  during development.The above analyses indicated that the major biological functions of Gon4l/Udu may be mediated through these interacting proteins. For example, Bap1, Dnmt1, and Thoc1 are highly associated with increased embryonic tissue cell apoptosis and prenatal lethality Table . These eges Fig. . We perfap1 Fig. a–d, dnmtmt1 Fig. e–h, thococ1 Fig. i–l, and y3a Fig. m–p. Surpy3a Fig. a,e,i,m, les Fig. c,g,k,o. hpf Fig. g,h,o,p. udu mRNA was expressed in a ubiquitous manner at early stages  and participates in DNA double-strand break (DSB) repair through regulating histone 2A and H2AX ubiquitylation38. Moreover, according to the online database BioGRID, Gon4l/Udu physically interacts with H2AX and other histone subtypes (Supplementary Table It has been demonstrated that DNA double-strand break (DSB)-induced DNA damage response (DDR) is activated in 39. It has been shown that lack of Dnmt1 expression leads to impaired self-renewal of hematopoietic stem cells (HSCs) in both adult mice and zebrafish models41. Down regulation of cebpa is crucial for the differentiation of pre-pro B cells to early pro-B cells44. In contrast to wild type, the mRNA level of cebpa is significantly up-regulated in the B cell progenitors of Justy mutant mice3. Further analysis using double morpholino injection revealed that Dnmt1 maintains HSCs and progenitor cells in zebrafish through regulating cebpa expression40. Similar defective hematopoietic phenotypes were also observed in Gon4l/udu mutants. The proliferation and differentiation of pre-pro B cells to early pro-B cells are impaired in Justy mutant mice4. In zebrafish, udutu24 mutants exhibited fewer blood cells and defective proliferation and differentiation of erythrocytes were observed in udusq3 mutants7. Although hematopoietic defects were not specifically described in the study using uduvu66 mutant embryos, its supplementary data using DamID-seq at the gastrula stage shows that the promoter region of cebpa was Udu-enriched, indicating that cebpa may be directly regulated by Udu8. Consistently, up-regulation of cebpa was observed in udutu24 mutants at 22 hpf  genes53. Also, it has been shown that Cry3a  is able to negatively regulate transcriptional activity of circadian transcription factor Clock1a:Bmal1b53, indicating conserved functions for those clock genes among species. Expression of cry3a mRNA has been identified in ventral telencephalon, retina, and intestine at 5 dpf32. In adult zebrafish, it is expressed in the brain, muscle, heart, and the liver31. Although only weak protein interaction signals between Udu and Cry3a  are involved in the circadian rhythms of plants and animals. In zebrafish, synchronization of circadian clock with cell cycle progression has been observed21. CRAMP1L and GON4L can also interact with MST1 when naïve CD4 + T cells differentiate into regulatory T cells (Treg) in another protein–protein interactome analysis17. Furthermore, Cramp1l is a polycomb group protein encoded gene that is dynamically regulated during cell cycle progression, peaks at S phase, and interacts with proliferating cell nuclear antigen (PCNA)55. In cramp1l mutants, the expression of histone H1, but not other histones, is greatly decreased54. Cramp1l has been shown to interact with another GON4L-interacting protein, NPAT 16. NPAT is a substrate for Cyclin E/CDK-2 and is responsible for biogenesis of histone locus body and histone gene transcription56. Therefore, Cramp1l and NPAT, together with Bap1, indicate that Gon4l/Udu may be essential for the connection between cell proliferation and histone expression/modification.In addition to the genes discussed above, Cramped-like (Cramp1l or Crm) has been shown to interact with Gon4l in a large-scale human interactome studyGon4l has been considered as a negative transcription regulator that inhibits gene expression probably through interacting with YY1, SIN3A, and HDAC1. In this study, we have identified four novel Gon4l/Udu-interacting proteins. These newly identified proteins not only confirm the association of Gon4l/Udu with histone biogenesis, as demonstrated in previous reports, but also indicate that Gon4l/Udu can possibly regulate both histone modifications and CpG DNA methylation to influence various cellular events. Furthermore, Gon4l may also maintain genomic integrity while controlling transcription and cell proliferation. Overall, these findings provide a basis for better comprehending the pleiotropic effects of Gon4l/Udu.The yeast two-hybrid (Y2H) assays for zebrafish Udu and mouse Gon4l were performed by Hybrigenics  and Myriad Genetics , respectively. Briefly, LexA and Gal4 system based on transcriptional activation of reporter genes was used to detect protein interactions. Bait fragments used for zebrafish Udu were the N-terminal CR1-CR3  and the C-terminal PAH and SANT domains . The corresponding fragments of zebrafish Udu were cloned, checked by sequencing, and used as bait to screen for protein interactions using zebrafish embryo RP1 library (stages 18–20 hpf). A total of 56 million and 82 million colonies for Udu  and Udu  were analyzed for the interaction, respectively. The resulting sequences were searched against GenBank using an automated procedure. Bait fragments used for mouse Gon4l were the two PAH domains  and SANT domain . The two fragments were cloned and used for protein interactions with prey libraries from murine embryos, B lymphocytes (B cells), and cardiac myocytes. Around 5–10 million colonies were obtained after each mating, then picked from the selection plates and the prey inserts were identified by sequence analysis. To confirm the interactions, the bait and prey plasmid DNAs were isolated and co-transformed into a naïve yeast strain to recapitulate the interaction.Danio rerio) were raised and kept under standard conditions according to a previous report57. Wild-type and udutu24 embryos were staged as described before58. All experimental procedures on zebrafish and their embryos were approved by the Institutional Animal Care and Use Committee of the National Health Research Institutes, Taiwan (NHRI-IACUC-106063-A) and carried out in accordance with the approved guidelines. Only zebrafish and no other animals were directly involved in this study.Zebrafish . One microgram of total RNA was used for subsequent reverse transcription using GoScript Reverse Transcriptase (Promega). The obtained cDNA was used for subsequent PCR cloning.udu C-terminally fused with Flag tag was PCR amplified. PCR product was subcloned into pcDNA3.1(+) at EcoRV/NotI sites. For the expression of PAH and SANT domains of Udu, pcDNA3.1(+)-flag-udu-P/S was used19. To perform co-IP analysis, candidate Udu-interacting genes were cloned from cDNA. For bnipl, brd4, cry3a, ephx2, foxa2, gc, kif20a and lias, sequence of Myc tag was added to their antisense primers. The PCR product of cry3a was digested with EcoRV/NotI and directly cloned into the corresponding sites of pcDNA3.1(+). The PCR products for bnipl, brd4, ephx2, foxa2, gc, kif20a and lias were first cloned into pJet1.2 vector and subcloned into pcDNA3.1(+) with NotI/XbaI. The PCR product for bap1 was first cloned into pJet1.2 vector and subcloned into pCS2 + MT vector with XhoI/XbaI. The PCR products for dnmt1 and thoc1 were first cloned into pJet1.2 vector and subcloned into pcDNA3.1(+)-MT vector with EcoR1/Not1 and Not1/Xba1, respectively. The accession numbers for these genes and their corresponding primer pairs used in this study are listed in Supplementary Table Full-length zebrafish 6 cells/well were seeded in a 6-well plate one day before transfection. One microgram of Myc-tagged constructs and/or one microgram Flag-tagged udu constructs were transfected into HEK293 cells using Lipofectamine 3,000 transfection reagent (Invitrogen). For co-IP analysis, 4 × 106 HEK293 cells or 2 × 106 COS-7 cells were seeded in a 60-mm dish one day before transfection. Two micrograms of Myc-tagged constructs and two micrograms Flag-tagged udu constructs were co-transfected. The pCS2-flag vector was used as a negative control for these experiments.HEK293 or COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). For immunofluorescent staining, 2 × 10To examine the expression pattern of Udu, Udu-P/S and all the interacting candidate proteins, immunofluorescent staining was performed 1 day after transfecting plasmids into HEK293 cells. After washed with PBS, fixed with 4% paraformaldehyde and permeabilized with PBST (0.3% TritonX-100), the cells were blocked with 5% BSA/PBST. Incubation with mouse anti-FLAG antibody  or rabbit anti-MYC antibody  at 4 °C overnight was followed by overnight incubation with Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 564-conjugated anti-rabbit secondary antibody (Molecular Probes). SlowFad Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) was used for nuclear counterstain and mounting. The fluorescent signals were taken by Axio Imager A1 (Zeiss) or TCS SP5 confocal microscope (Leica).Two days after transfection, cells were harvested and lysed with Pierce IP Lysis Buffer  with the addition of protease inhibitor cocktail . The lysates were clarified by centrifugation and determined for their concentration using Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 40 μl of anti-FLAG M2 magnetic beads (Sigma-Aldrich) was washed with lysis buffer and added to 800 μg of total protein lysates. After overnight incubation at 4 °C, supernatants were removed and the magnetic beads were washed in lysis buffer for 4 times. A total of 80 μl of co-IP elution buffer  was added to each sample. The samples were incubated with gentle shaking for 5 min at room temperature to elute precipitated proteins. Then the magnetic beads were spun down and the supernatants were transferred immediately to fresh Eppendorf vials containing 10 μl of neutralizing buffer . SDS-PAGE and subsequent Western blot analyses were performed with mouse anti-FLAG M2  or rabbit anti-MYC antibody . To enhance the signals, Western Blot Ag Signal Enhancer (Biomate) was used prior blocking the PVDF membranes with 5% skim milk (BD Difco) according to the manufacturer’s instructions. Mouse or rabbit secondary antibody conjugated with HRP  was applied. The chemiluminescent signals were visualized by Immobilon HRP substrate kit (Millipore) and captured by the BioSpectrumAC Imaging System (UVP). Full-length blots are included in Supplementary Fig. 28. modPhEA reports enriched phenotypes that are associated with a given group of genes. Therefore, modPhEA was used to analyze phenotypes enriched by Gon4l/Udu-interacting proteins. Twenty genes encoding for Gon4l/Udu-interacting proteins with mouse Ensembl IDs were input manually as dataset 1 and compared against the rest of the genes in the genome that represent the background (dataset 2). Enrichment analyses were performed based on phenotype databases from either mouse or Caenorhabditis elegans because of their most abundant results obtained after analyses. To calculate and compare the differences between these two groups of genes (set 1 and set 2), Fisher’s Exact Test with differentially enriched hypothesis was applied. The Benjamini–Hochberg Procedure was used for correction of the p-value to decrease the false-discovery rate.The model organism Phenotype Enrichment Analysis (modPhEA) is a freely available tool that collects phenotypic data from both mutagenesis and knockdown animal experiments among different species, including budding yeast, roundworm, fruit fly, zebrafish, mouse, and humanbap1 probe template preparation, an 842 bp fragment of bap1 C-terminal was PCR amplified from diluted pCS2-MT-bap1 plasmid DNA . For dnmt1 probe synthesis, a 3,197 bp fragment of dnmt1 was PCR-amplified from pcDNA3-MT-dnmt1 plasmid DNA . For thoc1 probe synthesis, we cut the ready-made pJet1.2-thoc1-antisense full-length plasmid with SacI. After enzyme digestion, a 649 nt thoc1 probe was synthesized by MEGAscript T7 Transcription kit. For cry3a probe synthesis, a 1865 bp fragment of cry3a was PCR-amplified from pcDNA3-cry3a-MT plasmid DNA . The bap1 and thoc1 probes were labeled with fluorescein labeling mix (Roche), while the udu probe was labeled with digoxigenin labeling mix (Roche). Detailed methods for probe synthesis and removal of template DNA were according to the manufacturer’s protocol. To examine the expression patterns of bap1, thoc1, and udu during zebrafish development, embryos/larvae were harvested at 3, 6, 22, 24, 36, 48, and 72 hpf. Whole mount in situ hybridization was performed as described previously59. Embryos were mounted in glycerol (Sigma) or Murray’s Clear , and images were taken by Zeiss Axiovision Imager A1 or Zeiss Discovery V8.For udutu24 embryos at 22 hpf as previously described. 1 μg of total RNA was reversely transcribed, and the cDNA was diluted and amplified with the respective primers listed in Supplementary Table gapdh was used as an internal control. The SYBR Green-based qPCR was carried out on Roche LC480 II using SensiFAST SYBR Hi-ROX Kit (Bioline). The program for qPCR was as follows: one cycle of 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 20 s. A program for melting curve was added to examine for the existence of primer dimers or non-specific signals. Data were expressed as fold-change compared to wild type sample (mean ± SD).RNA was extracted from wild type and Supplementary file1


Four patients received intravenous insulin with fasting and had a rapid reduction in triglyceride levels by 65–77% within 24 h; one patient had mild hypoglycemia secondary to therapy. Conclusion: HTGAP occurred in 4% of AP cases and was associated with higher risk of ICU admission. Intravenous insulin and fasting appear safe and efficacious for acutely lowering triglyceride levels in HTGAP.Background: Hypertriglyceridemia-associated acute pancreatitis (HTGAP) has been linked with increased severity and morbidity. In this study, triglyceride levels were measured in all patients admitted with acute pancreatitis (AP) to establish the incidence of HTGAP in an Australian center. Methods: A prospective cohort with AP was collated over an 18-month period in a single tertiary referral hospital. HTGAP was defined as AP with triglycerides ≥ 11.2 mmol/L (1000 mg/dL). Incidence, clinical co-morbidities, severity and management strategies were recorded. Results: Of the 292 episodes of AP, 248 (85%) had triglycerides measured and were included. HTGAP was diagnosed in 10 of 248 (4%) AP cases. Type 2 diabetes, obesity, alcohol misuse and gallstones were common cofactors. The HTGAP group demonstrated severe hypertriglyceridemia compared to the non-HTGAP group (median 51 mmol/L vs. 1.3 mmol/L). Intensive care unit (ICU) admissions were significantly increased (odds ratio (OR) 16; 95% CI 4–62) in the HTGAP group (5/10 vs. 14/238 admissions,  Hypertriglyceridemia-associated acute pancreatitis (HTGAP) is thought to account for 0.7–20% of acute pancreatitis (AP) cases and is associated with increased severity and morbidity ,7,8,9,10The potential mechanism of pancreatitis in hypertriglyceridemia remains unclear and largely based on animal studies ,14. HaveIf, indeed, hypertriglyceridemia is pathological in humans, whether and how to intervene remains unclear, with ongoing debate around the use of intravenous insulin infusion and lipid apheresis .This study prospectively examines the incidence of HTGAP in Australia in a single tertiary center, with a primary aim of routinely measuring triglyceride levels in order to establish incidence of HTGAP. A secondary aim was to describe a case series of patients with HTGAP with a focus on their co-morbidities, severity, and in-patient management.A prospective cohort was collated over 18 months (July 2017 to January 2019) in John Hunter Hospital, a tertiary referral hospital in New South Wales, Australia. This study received ethics approval from the Hunter New England Human Research Ethics Committee for low and negligible risk (Reference number 17/06/21/5.10). Patients presenting to this center with acute pancreatitis  and the Mann–Whitney test (non-parametric). Logistic regression was performed to estimate the association between HTGAP and ICU admissions. All two-sided p-values < 0.05 were considered significant.Statistical analyses were performed using STATA version 14. Demographic and clinical data were reported as a number with percentages for categorical variables, mean ± standard error for continuous variables that show a normal distribution and median with interquartile range (IQR) for continuous variables without a normal distribution. The comparisons for categorical data were performed using Fisher’s exact test, and continuous data comparisons were performed using the Student’s Of the 292 episodes of AP, 248 (85%) had triglycerides measured and were included in the cohort . HTGAP wn = 10), highlighting their clinical features, contributing factors and management. Within the HTGAP group, all cases had at least one secondary risk factor  (n = 6) and elevated body mass index  were common. Three HTGAP patients were already on lipid-lowering therapy prior to their acute admission, and two patients had previous admissions for HTGAP before July 2017. Alcohol misuse (n = 5) and gallstones (n = 3) may have directly contributed to the development of AP in the HTGAP group.obesity) . Type 2 p < 0.001) in the HTGAP group  and constituted 26% (5/19) of total ICU admissions for AP. APACHE II scores for those admitted to ICU were similar in HTGAP compared to non-HTGAP patients (median (IQR): 10 (5) vs. 16 (12), p = 0.266) (p = 0.382) . ICU admissions were significantly increased  . There w= 0.382) . There wn = 6, fibrate n = 5, omega-3 n = 1).The endocrine team was asked to provide a recommendation for the management of hypertriglyceridemia in seven of the patients. Four patients (three with diabetes and one without diabetes) received intravenous insulin infusion therapy, which, together with fasting, resulted in rapid improvement of triglyceride levels by 65–77% within 24 h of admission . These tThe only adverse event related to therapy was observed in Case 6, who had mild hypoglycemia (3.8 mmol/L) during intravenous insulin therapy  and was Case 9 was notable as they did not have a history of diabetes mellitus (HbA1c 5.6% or 38 mmol/mol) and was treated in a ward setting using fixed-rate intravenous insulin of 2 units/h (0.02 units/kg/h) administered concurrently with intravenous 4% dextrose + n/5 saline at 80 mL/h . The patWe report hypertriglyceridemia-associated acute pancreatitis (HTGAP) in 4% of acute pancreatitis cases in our regional tertiary referral center, with an increased rate of ICU admission in the group with severe hypertriglyceridemia. The link between hypertriglyceridemia (HTG) and acute pancreatitis was first observed by Speck in 1865 . DespiteSecondary causes of hypertriglyceridemia have been identified as common in the literature and were universally identified in the HTGAP individuals in this study. Secondary causes were similarly present in up to 46% of men and 94% of women with severe HTG (defined as >10 mmol/L) in the Copenhagen General Population Study (CPGS) and in 74% of cases in a Japanese study (defined as ≥11.2 mmol/) ,27. TypeIt is notable that seven of the reported HTGAP cases in this study had either a concomitant gallstone or high-risk alcohol intake . DominguSevere hypertriglyceridemia is associated with an increased severity of AP, a finding that has been demonstrated in both animal and human studies ,40,41,42The initial management of HTGAP is common to other forms of AP ,24. It in = 66 HTGAP) that compared high-volume hemofiltration (HVHF) against combination low-molecular-weight heparin (LMWH 4000 IU every 12 h for 3 days) and insulin (titrated via a pump to maintain blood glucose level between 7.8 and 11.1 mmol/L (140–200 mg/dL)) [The therapeutic options for acutely lowering triglyceride levels in HTGAP include IV insulin, heparin and lipid apheresis . Insulin mg/dL)) . HVHF ac mg/dL)) . However mg/dL)) .IV insulin therapy, when combined with fasting, is an effective management option as it has been shown to reduce triglyceride levels by 87% in 24 h, as compared to a 40% TG reduction with IV insulin alone, and 23% with subcutaneous insulin alone . Four paLipid apheresis rapidly reduces TG by 50–80% . HoweverThe main strength of our study is the prospective design that allows a reliable estimation of the incidence of HTGAP. Our incidence rate of 4% is much higher than the rate of 0.8% derived from an earlier observational audit performed retrospectively in the same center prior to the introduction of this study protocol . This caThe size of the HTGAP cohort and observational design of this study limit the ability to assess the efficacy of the management interventions for HTGAP. The limitations of an observational study led to some missing data points, particularly for HbA1c and body mass index. The HTGAP cohort contained few patients with HTG as a sole potential factor that could have contributed to the development of AP, and we were not able to perform logistic regression modelling to evaluate associated clinical risk factors for ICU admissions in HTGAP. This likely reflects ‘real life’, in which there are multiple triggers.HTGAP occurred in 4% of AP in a single Australian center and was associated with higher risk of ICU admission. Hypertriglyceridemia is a marker of severity for AP and could, in itself, be a causative factor. In our study, fasting, low-fat diet and intravenous insulin therapy were used to reduce triglyceride levels. At the present time, the use of IV insulin in patients without diabetes remains experimental , but it


The etiology of major neurodevelopmental disorders such as schizophrenia and autism is unclear, with evidence supporting a combination of genetic factors and environmental insults, including viral infection during pregnancy. Here we utilized a mouse model of maternal immune activation (MIA) with the viral mimic PolyI:C infection during early gestation. We investigated the transcriptional changes in the brains of mouse fetuses following MIA during the prenatal period, and evaluated the behavioral and biochemical changes in the adult brain. The results reveal an increase in RNA editing levels and dysregulation in brain development-related gene pathways in the fetal brains of MIA mice. These MIA-induced brain editing changes are not observed in adulthood, although MIA-induced behavioral deficits are observed. Taken together, our findings suggest that MIA induces transient dysregulation of RNA editing at a critical time in brain development. Interferon can be induced by the viral mimic PolyI:C, which can in turn activate RNA editing enzymes. Here the authors identify transient changes in A-to-I RNA editing following maternal immune activation in mice. Possible environmental risk factors for MND include intrauterine stressors, such as maternal bacterial and viral infections during pregnancy10.The etiology of major neurodevelopmental disorders (MND) such as schizophrenia and autism still remains a mystery. Current evidence suggests that a combination of polygenic susceptibility and exposure to environmental risk during sensitive periods of brain development can impair neurodevelopment, and confer vulnerability to the development of MND11. Infection-induced disruption of early fetal brain development was shown to significantly affect postnatal brain development and maturation, and to lead to structural and functional deficits that are dependent on postnatal maturational processes13. MIA models therefore possess both the construct and face validity to study MND, and are pivotal in the quest to elucidate the underlying biology responsible14. However, despite much progress, the biological mechanisms through which MIA exerts a long-term deleterious effect on brain development and function remain unknown.Maternal immune activation (MIA) rodent models that are based on this observation involve exposing a pregnant rodent to specific viral pathogens, immune-stimulating agents, or pro-inflammatory cytokines, and evaluating the subsequent long-term brain and behavioral effects in the offspring16. PolyI:C is a commercially available synthetic analog of double-stranded RNA, which is recognized by the mammalian immune system as foreign, due to its similarity to viral structures17. Systemic administration of PolyI:C leads to induction of the innate immune system, by activation of dsRNA sensors, such as melanoma differentiation-associated protein 5 (MDA5) and Toll-like receptor 3 (TLR3)18. These in turn, lead to the expression of innate immune response genes and proteins, as well as an induction of the interferons IFNα and IFNβ19.The viral mimic polyriboinosinic–polyribocytidilic acid (PolyI:C) has been shown to induce a multitude of behavioral, cognitive, and pharmacological abnormalities20, present both in the nucleus and in the cytoplasm21. The ADAR family of enzymes carry out the common post-transcriptional modification, A-to-I RNA editing, in which ADAR deaminases adenosine to inosine24. This editing is applied to long double-stranded RNA transcripts, which are recognized by MDA5 in the cytoplasm25, or to RNA elements in the nucleus. Since inosine is recognized by the ribosome as guanosine during translation, editing can alter the proteome outcome.Among its many biological effects, interferon (IFN) is responsible for the transcription of the Adenosine Deaminase Acting on RNA (ADAR) protein  in its IFN inducible form, the p150 isoform28, as well as in the genomes of many other organisms29. Alterations in editing levels are associated with a number of neurological disorders, including dementia and epilepsy31. The vast majority of A-to-I RNA editing activity occurs in non-coding primate-specific Alu repetitive elements35. Even though most of these sites occur in non-coding regions of the genome, they appear to be essential for development36. Notably, there is an enrichment of coding sites in neural tissues. These are predominantly edited by ADAR2 (refs. 38), and since many known editing sites are located within genes involved in brain function, their editing may also produce a phenotypic effect37. In spite of the fact that brain related lethality is more associated with deficiency of ADAR2 rather than ADAR1, both ADAR enzymes share editing activity, and may compete for certain substrates. As a corollary, each ADAR is able to compensate for a deficiency of the other and in a few cases can even boost the normal levels of editing at the other’s sites41. This tight interplay implies that alterations in ADAR1 activity may also be important for proper brain function and development.The magnitude of RNA editing by ADAR proteins is immense, with millions of sites already identified in the human genome44. Editing of dsRNA, a structure commonly formed by viruses, prevents binding of dsRNA sensors such as MAD5 (refs. 44), thus averting an IFN response that could severely damage host cells. Furthermore, MDA5 knockout rescues an ADAR1 editing-deficient phenotype, suggesting that the most relevant physiological function of ADAR1 is to specifically prevent aberrant activation of MDA5-mediated immune sensing44. This underscores the suggestion that it is imperative to achieve a balance between RNA editing at key sites, while preventing stimulation of the IFN pathway.Despite the possible involvement in brain development, probably the most important function of ADAR1-mediated editing of endogenous double-strand RNA (dsRNA) is to prevent an inappropriate cytosolic innate immune system response46, our results indicate that activation of the IFN pathway by dsRNA at a critical time in pregnancy leads to changes in gene expression as well as a vast global increase in RNA editing levels, which may produce a high mutation load in the developing fetus.In this context, the present study was designed to examine the relationship between immune activation during pregnancy, the mechanism of RNA editing in the fetus, and the long-term emergence of behavioral phenotypes associated with MND in the offspring. In contrast to previous studies in adults that detected minor or no differences in RNA editing levels between healthy and MND brains49 and therefore could potentially explain the observed alterations in brain development. As robust activation of ADAR may lead to errant editing at unknown sites, we hypothesize that MIA induces temporary changes in RNA editing patterns during a critical period of  brain development. These can be responsible for long-term deleterious effects on brain function in later life, even if they are not measurable by the time the behaviors are observed.The study also revealed the presence of tight regulation of coding editing sites during mouse brain development. The deviation from normal editing levels detected in the PolyI:C-treated mice may affect protein function18. The control group had eight females and five males while the PolyI:C group had seven females and seven males.In order to verify that indeed the mice we treated prenatally with PolyI:C showed MND-related behaviors, we conducted two behavioral experiments, the prepulse inhibition (PPI) and locomotor response to amphetamine tests. These are considered to be behavioral endophenotypes relevant for schizophreniaPPI: We assessed sensorimotor gating by the paradigm of PPI of the acoustic startle response, since reduced PPI has been proposed as a biomarker of schizophrenia50. In the PPI test, a weak prepulse stimulus suppresses the response to a subsequent startling pulse stimulus. Our PolyI:C-treated mice demonstrated a deficit in PPI, irrespective of gender  indicating that the level of percent PPI increased as the level of prepulse increased. The factor of gender and its interactions were not significant . However, the factor of treatment was highly significant in its own right , as well as the interaction with prepulse levels. Similar results were observed in gender-specific strata, with slightly less significance, probably due to smaller sample sizes, leading to lower statistical power , with the between-subject effects of sex and prenatal treatment as well as 12 5-min time intervals, demonstrating a significant effect of treatment.The systemic administration of amphetamine  resulted in a general increase in locomotor activity. This response was enhanced in PolyI:C offspring Fig. , as reveF = 2.56, DF = 11/253, p < 0.005) as can be seen in Fig. A significant three-way interaction of sex × treatment × time intervals  <0.05, and revealed extensive changes in transcription  fetuses and 8 fetuses  from PolyI:C-treated mice at GD10 (see “Methods”). The results were analyzed for differential gene expression using thresholds of |logion Fig. . Gene SeAlu Editing Index (AEI) method55, which provides information about the general level of editing in intronic Alu. Instead of human Alu, we supplied the AEI with mouse genomic Short Interspersed Nuclear Element (SINE) regions, B1, where most of the editing activity in mouse occurs56. This level can be compared across samples to give an indication of RNA editing activity, even in cases where only low sequencing coverage is available55.In addition to gene expression analysis, we evaluated the global levels of A-to-I RNA editing directly from transcriptomic data using the The AEI in all B1 regions revealed a significant elevation in global editing in PolyI:C-treated mice compared to that of controls Fig. . Since P57, as an additional method to identify clusters of editing sites that are overlooked by standard alignment methods (see “Methods”). Here too, the results revealed a significant elevation of global editing in PolyI:C-treated mice compared to controls  identified in the hyperediting analysis correlated well with the AEI in each sample Fig. In order to further analyze the hyperediting output in each group we combined the A-to-G sites. When a site appeared in both the control and the PolyI:C treatment groups, it was classified as a “common site”.Intersecting the sites in each group with the refseq genes table, we could conclude that the total number of unique editing sites inside genes in the PolyI:C-treated group was 3 times the number of sites in the control group . We calculated the AEI in 138 , 115 , and 80  brain samples originated from the Cerebellum, Frontal Cortex, and Amygdala, respectively. Here too, no differences in editing levels could be associated with gender in any of the datasets  portal  rather than bona fide editing. The editing index expected to be the second highest after A-to-G is C-to-T, which represents both a common genomic mutation and the possibility of C-to-U RNA editing, and is therefore useful to estimate the signal-to-noise ratio.58 , we could verify the effect on editing changes by analyzing the original data. A comparison of the editing in PolyI:C and control mice yielded the exact same statistical power , with 220M reads per sample, and calculated the editing levels. A similar increase in levels of editing was found in both the original and the deep sequencing samples. Specifically, AEI Fig.  and hype21. Since most of the changes in editing occur in introns located in the nucleus, it was important to ascertain that the changes in editing we detected originate from ADAR-p150 activity. The results indicated that PolyI:C exposure markedly increased mRNA expression of ADAR1-p150, the IFN induced isoform20, with no significant alterations in the expression levels of ADAR1-p110 isoform detected  (see “Methods”). The sort of viral infection, simulated by PolyI:C, activates an IFN response, which via an IFN-inducible promoter, can lead to overexpression of the ADAR1-p150 isoform and subsequently to increased levels of RNA editing. In this case, it was important to examine whether the PolyI:C treatment indeed caused increased expression of the IFN-inducible ADAR1 isoform. As already discussed, the ADAR1 isoforms have different expression patterns, with ADAR-p110 located exclusively in the nucleus while ADAR-p150 is present both in the nucleus and in the cytoplasmTaken together, these results verify the A-to-I RNA editing origin of the observed A-to-G mismatches.59 to assess RNA editing level changes in coding sequences. Although millions of years of evolution separate humans and mice, these sites were found to be conserved within the mammalian lineage, and are thus presumed to have an important biological function60.RNA editing sites in coding sequences represent only a small, albeit important, fraction of the editing activity. We used a set of 59 known, evolutionarily conserved, editing sitesMany of the editing sites are situated in genes with low levels of expression during development and reliable editing levels could not be determined. However, ten conserved specific editing sites that passed the reads coverage cutoff (see “Methods”) were all found to have significantly increased levels of RNA editing in the PolyI:C-treated group, particularly the sites in Dact3, Cog3, and Blcap genes Fig. . NotablyThere were only minimal variations in the gene expression of the genes harboring the 59 conserved editing sites when comparing the PolyI:C and control groups, indicating that differential editing was unlikely to be secondary to alterations in gene expression Fig. .Further analysis of the deep sequenced samples showed a similar increase in editing levels in coding sites Fig. . This an41. Six out of the seven differentially edited sites were substrates for both ADARs  nine PolyI:C and eight control frontal cortex samples from mice subjected to MIA treatment at GD12.5 and sequenced on PD189. (2) 10 PolyI:C and 10 control amygdala samples from mice subjected to MIA treatment at GD9 and sequenced at the age of 12 weeks.As expected, the datasets exhibited no differences between PolyI:C-treated mice and the controls in either the levels of editing in coding sites Fig.  or in th61 three to four samples of fetal mouse brain at every developmental day, from embryonic day 10–18. The results were used to track the changes in A-to-I RNA editing levels in conserved coding sites59 and to calculate the AEI. This analysis revealed that the RNA editing levels62 of the various coding sites over the tested period of embryonic development were highly reproducible between samples  are associated with distinct transcriptomic changes in the prenatal period. RNA analysis of fetal brains in the period following the MIA led to several compelling insights and revealed extensive dysregulation in gene sets related to brain and neuronal development. To our knowledge, this is the first time that RNA-seq has been performed during this time period of mouse development following MIA, as most gene expression studies have considered only the adult offspring of MIA69 revealed changes in RNA editing in coding sites in cortical samples from adult individuals with schizophrenia. Since PolyI:C is an inducer of IFN, which promotes ADAR1-p150 expression20, it was of interest to consider whether changes in A-to-I RNA editing also play a role in the development of this disease. As predicted, we identified extensive changes in A-to-I RNA editing following MIA, with large differences in global editing levels between the PolyI:C-treated group and the control group, where the offspring of PolyI:C-treated mice exhibited much higher levels of editing overall.A recent study by Breen et al.59. Even though most of the functional changes caused by this editing are not yet known, the conservation of these sites over tens of millions of years speaks to their importance. RNA editing in conserved sites usually leads to amino acid substitutions. Here, we detected a general increase in editing in the mice treated with PolyI:C, and particularly in the recoding sites within the DACT3, COG3, and BLCAP genes. These are particularly interesting in that the Dact3 gene is essential for normal brain development, and important in embryonic development of the central nervous system70, while COG3, a part of the COG complex, interacts with Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1), which contains the schizophrenia susceptibility factor dysbindin. Dysbindin expression was previously shown to be reduced in the hippocampi and cortical areas of schizophrenia patients, which implicates the protein in molecular pathways leading to schizophrenia75.The A-to-I editing changes were investigated further by examining changes at specific editing sites, specifically a set of 59 editing sites in coding sequences known to be conserved in mammalian evolution76. Deviations in editing at the GRIA2 gene can lead to severe disease phenotypes78. Moreover, editing levels of GRIA2 were previously reported to be altered in postmortem brains from schizophrenia patients80. Analysis of hyperediting, which identifies clusters of editing sites that are overlooked by standard alignment, led us to conclude that there were significantly more editing sites inside genes in the PolyI:C group compared to control. While most of these sites were situated inside introns and therefore do not alter functionality, they may have an effect by changing the biogenesis of circRNA, alter the splicing pattern of the pre-mRNA or otherwise influence the expression of mRNA by editing the miRNA binding sites81.Since sequencing depth is crucial to confirm editing levels, we repeated the experiment on one sample from each group, with a much deeper coverage of 220M reads. Importantly, not only did the alterations in RNA editing become more robust, but the deep sequencing also revealed additional differentially edited sites in coding sequences. These included a Q/R GRIA2 site, which is a crucial site for editing in humans and miceSince all these changes occur at the level of a single cell, the overall effect may be slight and difficult to detect. However, if they take place at a critical time in development, they may be enough to cause a meaningful impact. It is important to remember that these changes are temporary and may be undetectable when the RNA or protein composition of an adult brain is analyzed.84, and detected no significant differences in overall editing levels compared to control samples . Litter mates of the same sex were kept in groups of three to five mice, and were maintained in standard conditions of constant temperature (22 ± 1 °C), humidity , and on an ad libitum food and water diet under a 12:12 light–dark cycle. All experimental protocols were authorized by the Tel Aviv University Committee of Animal Use for Research and Education.Breeding began after 7 days of acclimatization to the new animal holding room. Groups of 2–3 females were moved to separate cages with some urine-soiled wood chip bedding from a cage containing male C57BL6/J mice. On the fourth day of exposure to male urine, females were exposed to one male and allowed to mate. The next morning, the male mouse was removed from the cage, and the females were weighed. This day was referred to as gestational day (GD) 0. Females who showed an increase of over 2 g in body weight after 9 days were considered pregnant.We used two cohorts of offspring: (1) mice that were prenatally sacrificed soon after the MIA procedure to allow an examination of the short-term effects of MIA on RNA editing patterns in the brain  and (2) mice that were used to study the long-term behavioral effects of prenatal MIA .PolyI:C sodium  was dissolved in sterile 0.9% saline on the day of injection. Injections were made via the intravenous route into the tail vein under mild physical constraint, and the volume of injection was 5 ml/kg. The animals were returned to the home cage post-injection.15, which suggested that MIA in early pregnancy can lead to behavioral and cognitive dysfunctions relating to schizophrenia in the offspring.On gestational day 9, pregnant C57BL/6J dams were injected with PolyI:C (5 mg/kg/ml) or vehicle  into the tail vein. The timing of injection was based upon the work of Feldon and MeyerThe group of pregnant mice designated for RNA analysis of the fetuses were killed by decapitation 24 h after PolyI:C or vehicle treatment . The abdominal cavity was exposed, and the uterus was removed and placed in a Petri dish filled with ice-cold phosphate-buffered saline (PBS). The uterus was then transferred to another Petri dish containing ice-cold PBS and dissected under visual guidance using a dissecting microscope.RNA save solution  in an Eppendorf tube and frozen immediately by immersion in liquid nitrogen. Samples were stored at −80 °C until RNA extraction.The separated fetal heads were immediately placed in 89 in animals from the control group and healthy brain samples61, sequenced on the same developmental day, confirmed that the tissue originated mainly from the brain  was used to measure startle reactivity. The startle chamber comprised a nonrestrictive cylindrical enclosure made of clear Plexiglas attached horizontally on a mobile platform, which in turn rested on a solid base inside a sound-attenuated isolation cubicle. A high-frequency loudspeaker mounted directly above the animal enclosure inside each cubicle produced a continuous background noise of 65 dB  and various acoustic stimuli in the form of white noise.For the PPI testFor PPI of the acoustic startle reflex, subjects were presented with four types of trials. These were pulse-alone trials, prepulse-plus-pulse trials, prepulse-alone trials, and trials in which no discrete stimulus, other than the constant background noise, was presented . A reduction of the magnitude of the startle after prepulse-plus-pulse trials relative to that obtained after pulse-alone constitutes PPI. The pulse stimulus employed was 120 dBA in intensity and 40 ms in duration. Prepulses of various intensities: 69, 73, 77, 81, and 85 dB, corresponding respectively to 4, 8, 12, 16, and 20 dB above background, were employed. The duration of prepulse stimuli was 20 ms. The SOA (stimulus onset asynchrony) of the prepulse and pulse stimuli in the prepulse-plus-pulse trials was 100 ms.A session began with the animals being placed in the Plexiglas enclosure and allowed to acclimatize to the apparatus for 2 min before the first trial. The first six trials consisted of startle-alone trials, which served to habituate and stabilize the animals’ startle response. Subsequently, the animals were presented with 12 blocks of discrete test trials. Each block consisted of one trial of each of the following trial types: pulse-alone, prepulse-plus-pulse trials of each of the five levels of prepulse, prepulse-alone of each of the five levels of prepulse, and no stimulus . The session was concluded with a final block of six consecutive startle-alone trials. The interval between successive trials was variable with a mean of 15 s (ranging from 10 to 20 s).%PPI was calculated as /mean startle response to 120 dB pulse alone) × 100.d-amphetamine (Amphetamine Session 1). Thus, all animals served as their own controls.To investigate the behavioral activation induced by an acute amphetamine injection, all mice were tested in an open-field test composed of three sessions. First, spontaneous locomotor activity of the mice in response to novelty was recorded during 30 min without any drug treatment (Habituation Session 1). After this, all animals received an intraperitoneal injection of saline  and were returned immediately to the open-field arena for a further 30-min session . Finally, the locomotor activity response of the mice to an acute amphetamine treatment was recorded for a further 60-min after an intraperitoneal injection of 2.5 mg/kg A260:A280 and A260:A230 were measured. The integrity of RNA was evaluated based on RIN acquired via capillary gel electrophoresis performed using Agilent 4200 TapeStation in combination with Agilent RNA ScreenTape System . All RNA samples went through DNase Treatment Kit  before proceeding to the next step.Total RNA was extracted using the RNeasy Mini kit . Isolated and purified total RNA was then quantified using a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and the absorbance ratios For this particular study, we prepared a library of 19 RNA samples using NEBNext RNA ultra II RNA library preparation kit (E7770L). The input amount of total RNA was 200 ng which was in the recommended range. All RNA samples underwent PolyA selection following the manufacturers’ protocols.Samples were multiplexed using suitable molecular barcodes and resulting cDNA pools were processed according to the NextSeq System Denature and Dilute Libraries Guide. Firstly, the concentration of libraries was measured using a Qubit Fluorometer in combination with the Qubit dsDNA HS Assay Kit . Secondly, libraries were analyzed by Agilent 4200 TapeStation in combination with Agilent High Sensitivity D1000 ScreenTape System  according to the manufacturer’s protocol.Paired-read sequencing of the libraries with a read length of 150 was performed with NextSeq 500 Sequencing System using NextSeq 500/550 High Output v2 kit (20024906 Illumina). PhiX Control v3 (Illumina) was added at 1% to all pools as an internal control before sequencing.n = 7 for the control mice and n = 5 for the PolyI:C group, since not enough DNA was available from this group. Specific primers were used to amplify ADAR-p110 and ADAR-p150, and β-actin was included in each experiment as a loading control. The fold change in expression was determined using the ΔCt method. The following primer sequences were used for PCR—ADAR-p110 Forward Primer (FP): 5′-GCAGCGTCCGAGGAATCG-3′, Reverse Primer (RP): 5′-TAAGACTCCGGCCCCTGTG-3′; ADAR-p150 FP: 5′-CACTATGTCTCAAGGGTTCAGGG-3′, ADAR-p150 RP: 5′-CACTTGCTATGCTCATGACTAGGG-3′; and β-actin FP: 5′-AGAGCATAGCCCTCGTAGAT-3′, β-actin RP: 5′-CCCAGAGCAAGAGAGGTATC-3′.Two hundred and fifty nanograms of RNA were used in a reverse transcription reaction using the Verso cDNA Synthesis Kit . The cDNA obtained was used in real‐time PCR. Platinum SYBR GreenqPCR SuperMix-UDG w/ROX  was used to quantify gene expression. The number of mice used for this experiment was http://prinseq.sourceforge.net/). An average of 7% of the reads were removed from each sample .In RNA-seq data, duplicate reads bias, defined as reads that are identical and on the same strand, can occur and may affect the accuracy of analysis. These reads can be produced by the PCR cycles conducted before sequencing. To reduce sequencing biases, duplicate reads were removed using the PrinseqLite script version 0.20.4 (90).Transcriptomic data quality was evaluated using the FastQC quality control tool version 0.10.1 .The reads were mapped to the mouse reference genome version GRCm38/mm10 using STAR aligner version 2.5.2b56). To measure the editing in B1 elements, we used the AEI method version 1.0 (refs. 55).The most highly edited mobile element in the mouse genome is B1 , minimum 0.001 editing frequency (−n 0.001), exploring one base near the splice junction (−r 1), minimum one read coverage, and trimming of five bases at both ends of the reads (−T 5-5).For reliable editing evaluation, we removed sites which had less than 10 reads of alignment coverage. We also removed sites where less than 50% of the group’s samples showed editing signals. The statistical significance of differences between two groups was tested using the Wilcoxon rank-sum test with default parameters, followed by the Benjamini–Hochberg FDR multiple testing (0.1).93 following “tximport” R library version 1.12.3 (ref. 94) with default parameters and mouse genome version GRCm38/mm10. Principal components were computed for each sample using log2 (TPM + 0.1) of all genes. The prcomp R function was used. Differential gene expression was evaluated on Salmon transcript counts using DESeq95. For GSEA97 genes were transformed into their human homolog and ranked by multiplication of p value by log2 fold change for each gene. Mouse genes corresponding to more than one human gene were included multiple times with the same value. The pre-ranked option was used with the GSEA software version 4.0.2. Clustering of gene sets was performed in Cytoscape (version 3.4)98 using the Enrichment map module with default values. Clusters with >5 nodes (i.e. gene sets) were included in the plot.Gene expression levels in units of transcripts per million (TPM) were computed using the Salmon tool (version 0.11.2)The sex of each mouse fetus was determined by analyzing the gene expression levels of chromosome Y-linked genes DDX3Y and UTY. A threshold of expression levels in TPM units <1 was set to define female mice .The statistical calculations on the data and figure drawing were made using Rstudio, an integrated development environment for R , unequal sample sizes in the treatment factor, and same mouse exposed to multiple noise levels or measured on several time bins (repeated measures), we used linear mixed models with random mouse effects, and sex, treatment group, time interval/noise level as fixed effects, with distance/%PPI as outcome, followed by analysis of variance. Statistical analysis was performed using the publicly available R package lme4. Datasets, code, and reproducible reports of statistical analyses of the behavioral experiments are provided in Supplementary Notes Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting Summary


Because of this pathway, acetogens have been considered as a novel platform to produce biochemicals from gaseous feedstocks, potentially replacing the conventional thermochemical processes. Despite their advantages, a lack of systematic understanding of the transcriptional and translational regulation in acetogens during autotrophic growth limits the rational strain design to produce the desired products. To overcome this problem, we presented RNA sequencing and ribosome profiling data of four acetogens cultivated under heterotrophic and autotrophic conditions, providing data on genome-scale transcriptional and translational responses of acetogens during CO2 fixation. These data facilitate the discovery of regulatory elements embedded in their genomes, which could be utilised to engineer strains to achieve better growth and productivity. We anticipate that these data will expand our understanding of the processes of CO2 fixation and will help in the designing of strains for the desired biochemical production.Acetogens are anaerobic bacteria that utilise gaseous feedstocks such as carbon monoxide (CO) and carbon dioxide (CO Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13625942 The demand for sustainable replacements for fossil fuels to obtain environmental and economic sustainability has, therefore, also been increasing. Microbial gas fermentation has recently drawn much attention as an alternative method to convert gaseous feedstocks (CO or CO2) into biofuels and biochemicals, owing to its low operation cost compared to the conventional thermochemical process6. Among the organisms capable of gas fermentation, acetogenic bacteria (acetogens) are considered one of the most promising potential platforms8.Over the past decades, the usage of fossil fuels, which is now responsible for 40% of carbon dioxide  using one molecule of ATP. Subsequently, methenyl-THF, methylene-THF, and methyl-THF are synthesised using two additional reduction powers, which vary according to the acetogen. Subsequently, the methyl group of methyl-THF is transferred to the CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex to form acetyl-CoA, combining with the reduced CO and coenzyme A (CoA). The synthesised acetyl-CoA can be converted into acetate to recoup ATP or biomass building blocks.A unique feature of acetogens is the presence of the Wood-Ljungdahl pathway (WLP) coding genes in their genomes+ oxidoreductase (Rnf) and the Fd: H+ oxidoreductase (Ech) complexes to translocate ions across the membrane to create ion gradient14. The established gradient drives the ion back into the cell through the ATP synthase complex, generating the ATP needed for the cell. Along with the ATP synthesis system, electron bifurcation, which oxidises one electron donor and transfers electrons to two different electron acceptors, helps to overcome energetic barriers by reducing low reduction potential Fd via oxidisation of a relatively higher reduction potential hydrogen molecule, which can then reduce CO2 and the ion translocating complex16.In addition to the WLP, energy conservation systems play essential roles in acetogens by compensating for the energy required for biomass synthesis. These systems utilise the ferredoxin (Fd): NADAlong with the WLP and the energy conservation system, acetogens, similar to any other organism, contain intricate regulatory networks to control gene expression under different conditions. To date, a large number of acetogens have been sequenced to identify their genomic features; this further required a systematic understanding of their transcriptional and translational regulatory processes. In contrast to genomic studies, relatively few studies on acetogens’ transcriptomes and translatomes under autotrophic growth conditions have been published. The lack of uniformly generated RNA sequencing (RNA-Seq) and ribosome profiling (Ribo-Seq) data for acetogens has not only limited from obtaining knowledge on the cellular responses but also expanding potential genetic tools for strain engineering. RNA-Seq and Ribo-Seq can determine the strength of promoters and Shine-Dalgarno (SD) sequences in the 5´-untranslated regions, which regulate transcription and translation, respectively.Acetobacterium woodii, Clostridium aceticum, Clostridium drakei, and Eubacterium limosum cultured under the two conditions. Although studies on E. limosum and a transcriptomic study on C. drakei have been described in previous studies by our group, this study provides a uniformly generated and processed dataset of the additional model acetogens, which allows the comparative analysis of the transcriptome and translatome of CO2 fixing acetogens18. The presented RNA-Seq and Ribo-Seq results will provide a fundamental understanding of the responses of the acetogens to autotrophic conditions, and thereby widen genetic tools for strain engineering to produce biochemicals using CO2 as a carbon building block.In this study, we determined changes in the transcriptional and translational responses of acetogens under autotrophic growth condition compared to heterotrophic growth conditions, using RNA-Seq and Ribo-Seq. RNA-Seq and Ribo-Seq were performed on four acetogen species, A. woodii DSM 1030, C. aceticum DSM 1496, C. drakei DSM 12750, and E. limosum DSM 20543 were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures . A. woodii, C. aceticum, and C. drakei were cultured under strict anaerobic conditions at 30 °C and E. limosum was cultured under anaerobic conditions at 37 °C in 150 mL serum bottles filled with 100 mL DSMZ 135 medium (pH 7.0), which is composed of 1 g L−1 NH4Cl, 2 g L−1 yeast extract, 10 g L−1 NaHCO3, 0.1 g L−1 MgSO4 × 7H2O, 0.3 g L−1 cysteine-HCl, 10 mL vitamin solution , 20 mL trace element solution 2 × 12H2O, 10 mg L−1 H3BO3, 10 mg L−1 Na2MO4 × 2H2O, 30 mg L−1 NiCl2 × 6H2O, 0.3 mg L−1 Na2SeO3 × 5 H2O, 0.4 mg L−1 Na2WO4 × 2H2O), 4.6 mM KH2PO4, 5.4 mM K2HPO4, and 4 µM resazurin. For heterotrophic growth, A. woodii, C. aceticum, and C. drakei were cultivated in the fructose supplemented (5 g L−1) media and E. limosum was cultured in glucose supplemented (5 g L−1) media. For autotrophic growth, all of the strains were cultivated in DSMZ 135 media containing H2/CO2 (80:20) with pressure of 200 kPa in the headspace (50 mL). The media used for culturing A. woodii was supplemented with 2 g L−1 NaCl. For the main culture, the precultured cells were harvested via anaerobic centrifugation, then washed with basal DSMZ 135 media three times and inoculated in 100 mL fresh DSMZ 135 media supplemented with corresponding carbons. All of the strains were cultured in biological duplicates.For this study, g for 15 min at 4 °C. The collected cells were resuspended anaerobically in 500 µL of lysis buffer, comprising 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Liquid nitrogen was used to freeze the samples, which were then ground using a mortar and pestle. Lysed cells were thawed on ice, and the debris was removed by centrifugation at 4,000 g for 15 min at 4 °C. Subsequently, the total RNA was isolated using TRIzol  according to the manufacturer’s instruction. To remove the remaining genomic DNA (gDNA), the RNA was treated with 4 U of rDNase I  for 1 h at 37 °C, then incubated at 75 °C for 10 min to deactivate the enzyme. To remove ribosomal RNAs (rRNA) in the gDNA-depleted RNA, the Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria  was used according to the manufacturer’s instruction. The quality of the rRNA-depleted RNA was checked using an Agilent 2200 TapeStation system . To construct the libraries for RNA-seq, the TrueSeq Stranded mRNA Library Prep Kit  was used on the quality confirmed RNA. The libraries were sequenced using the 150 bp read recipe with an Illumina MiSeqTM system.Duplicate samples were harvested at the mid-exponential phase by centrifugation at 4,000 2, and 100 µM CM, and resuspended in lysis buffer consisting of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, 100 µM CM, and 1% Triton X-100. The resuspended cells were frozen in liquid nitrogen and ground with a pestle and mortar. The powdered cells were recovered by centrifugation at 4,000 g for 15 min at 4 °C, and the resultant supernatant was additionally centrifuged at 16,000 g for 10 min at 4 °C. To degrade RNA unprotected by ribosomes, 400 U MNase , 2 µL bovine serum albumin (1 mg mL−1), and 20 µL of 10 × MNase buffer were added and samples were incubated at 37 °C for 2 h with gentle rotation. To inactivate the reaction, 10 µL 0.5 M EGTA  was added to the sample. The monosome fraction was recovered using Microspin S-400 HR columns . The recovered ribosome-bound RNA was isolated using TRIzol, and the remaining rRNAs were removed with the Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria. For the phosphorylation reaction, samples were denatured at 80 °C for 90 s, equilibrated to 37 °C, and incubated at 37 °C for 1 h with 5 µL 10 × T4 PNK buffer (NEB), 20 U SUPERase-In RNase Inhibitor, and 10 U T4 PNK (NEB). After purification of the RNA samples using RNeasy MinElute Column , the concentration of purified RNA was measured using the Qubit RNA HS assay kit . For library construction, the small RNA library prep kit for Illumina (NEB) was used, and the constructed library was sequenced using the 50 bp read recipe on an Illumina Hiseq2500.For Ribo-Seq, 100 µM chloramphenicol (CM) was added to the cultures which were then further incubated at 30 °C or 37 °C, corresponding to the duplicate culture conditions, for 10 min. The CM treated cells were subsequently washed using 500 µL polysome buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgClA. woodii (NC_016894), C. aceticum (NZ_CP009687), C. drakei (NZ_CP020953), and E. limosum (NZ_CP019962) genomes using default parameters  and only the uniquely mapped reads were rescued. The gene expression was calculated from the mapped read count statistics using the DESeq. 2 package in R with default parameters. For Ribo-Seq, the adaptors of the generated reads and quality below Phred score of 20 were removed, then trimmed with the same parameters applied for RNA-Seq, and reads shorter than 20 bp were again removed. The reads were mapped onto the A. woodii (NC_016894), C. aceticum (NZ_CP009687), C. drakei (NZ_CP020953), and E. limosum (NZ_CP019962) genomes using the default parameters  and only the uniquely mapped reads were rescued. Using the obtained reads, the gene expression was normalised using the DESeq. 2 package in R with default parameters.For RNA-Seq, the adapter sequence of the sequenced reads and quality below Phred score of 20 were trimmed. Trimmed reads shorter than 20 bp were discarded to improve the accuracy of the mapping result. Using CLC Genomics Workbench , the trimmed reads were mapped onto the A. woodii were deposited in the European Nucleotide Archive under study accession PRJEB3346019. The RNA-Seq and Ribo-Seq datasets for C. aceticum were deposited in the European Nucleotide Archive under study accession PRJEB3613420. The RNA-Seq and Ribo-Seq datasets for C. drakei, published in previous study18, were deposited in the NCBI Gene Expression Omnibus (GEO) repository with the accession code GSE118519 and the European Nucleotide Archive under study accession PRJEB3613522. The RNA-Seq and Ribo-Seq datasets for E. limosum, published in previous study, were deposited in the NCBI Gene Expression Omnibus (GEO) repository with the accession code GSE9761323. Detailed information on analysis of RNA-Seq and Ribo-Seq has been deposited in Figshare24.The RNA-Seq and Ribo-Seq datasets for 2 using the efficient WLP and the energy conservation system. To systematically understand this metabolism, four acetogen species, A. woodii, C. aceticum, C. drakei, and E. limosum were cultivated under heterotrophic or autotrophic condition, then sampled at the corresponding mid-exponential point . For RNA-Seq, the total number of bases mapped to on Table  and prinon Table , which von Table . Subsequely Fig. .Fig. 2QuA. woodii, C. aceticum, C. drakei, and E. limosum, respectively (Fig. Similar to the RNA-Seq data, the Ribo-Seq reads were normalised using DESeq. 2, and the reproducibility of the replicates was confirmed via hierarchical clustering Fig. . Followiely Fig. .