The psychosis risk factor RBM12 encodes a novel repressor of GPCR/cAMP signal transduction

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RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293 cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cyclic AMP/protein kinase A (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR/cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.

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  1. Review coordinated by Life Science Editors Foundation

    Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation

    Potential Conflicts of Interest: None

    Punch line: Rare risk variants associated with schizophrenia converge on the cAMP/PKA pathway.

    Why is this interesting? The cAMP/PKA pathway could be a mechanism & therapeutic target for neuropsychiatric disorders arising from different mutations.


    • About 1-4% of people will develop psychosis or schizophrenia.
    • Schizophrenia is a highly heritable disease.
    • Genetic loci associated with schizophrenia can be common variants, which typically have small effects on risk, or rare variants, which can have large effects.
    • Rare, protein-truncating variants substantially increase the risk for mental illnesses like schizophrenia.
    • Disease-associated genes have diverse functions (e.g.):
    1. RNA binding (RBM12)
    2. transcriptional regulation (SP4, RB1CC1, SETD1A)
    3. splicing (SRRM2)
    4. signaling (AKAP11)
    5. ion transport (CACNA1G, GRIN2A, GRIA3)
    6. neuronal migration and growth (TRIO)
    7. nuclear transport (XPO7)
    8. ubiquitin ligation (CUL1, HERC1) ** What are the pathological mechanisms?*
    • A genetic screen identified the risk gene RBM12 as a novel repressor of GPCR/cAMP signaling (Semesta et al., PLOS Genetics, 2020).
    • Dysregulation of GPCR activity in the brain contributes to the pathophysiology of several neurological and neuropsychiatric disorders.
    • cAMP is a critical second messenger that mediates all important aspects of neuronal function, including development, excitability, and plasticity.


    • Use knockout HEK293 cells to verify that RBM12 is novel repressor of the GPCR/cAMP pathway that extends to multiple GPCRs coupled to the stimulatory G protein (e.g. dopamine 1 receptor, beta-2 adrenergic receptor).
    • Show RBM12 also represses this pathway in iPSC-derived neurons. RBM12 knockdown yielded hyperactive upregulation of NR4A1 and FOS mRNAs, two known CREB-dependent immediate early genes induced by neuronal activity.
    • RBM12 loss leads to increased PKA activity and supraphysiological CREB-dependent transcriptional responses.
    • RBM12 loss increased expression of the endogenous β2-AR transcriptional target mRNAs, PCK1 and FOS.
    • RBM12 loss increased CREB transcriptional reporter expression in response to a panel of endogenous or synthetic β2-AR agonists.
    • Transcriptional responses are orchestrated from endosomal β2-ARs in wild-type cells but from both plasma membrane and endosomal β2-ARs in RBM12 knockout cells.
    • Their results suggest that cAMP production and transcriptional signaling are independently subject to RBM12 regulation.
    • The neuropsychiatric disease-linked mutations fail to rescue GPCR-dependent hyperactivation in cells depleted of RBM12.
    • Defined β2-AR-dependent transcriptional targets in “wild-type” and RBM12 knockdown neurons by differential expression analysis between each respective basal and isoproterenol conditions. 669 unique β2-AR-dependent transcriptional targets across the two cell lines.
    • Discerned β2-AR-dependent targets that were exclusive to wild-type or RBM12 knockdown only (qualitatively distinct targets) versus targets that are in wt and RBM12 kd but upregulated to different extents (quantitatively distinct targets).
    • 21 wild-type- and 115 RBM12 knockdown-specific target genes. Factors involved in synaptic plasticity and schizophrenia such as JUN, ARC (encoding the activity-regulated cytoskeleton-associated protein), BDNF, and NRXN3 (encoding the cell adhesion molecule neurexin-3-alpha) were induced by GPCR signaling only in RBM12 knockdown neurons, while GRIA2 (encoding the AMPA receptor) and CBLN2 (encoding cerebellin 2 precursor) were upregulated upon GPCR signaling only in wt neurons.
    • the remaining 533 genes were induced in both wt & RBM12-depeleted, with a trend toward RBM12-dependent hyperactivation.
    • loss of RBM12 leads to aberrant expression of ADCY, PDE, and PRKACA, suggesting this mechanism underlies the hyperactive GPCR/cAMP/PKA signaling phenotypes.


    • Dysregulation of GPCR signaling could contribute to the neuronal pathologies stemming from loss of RBM12.
    • RBM12 function is required for normal cAMP production downstream of many Gαs-coupled receptors with established roles in the nervous system consistent with dysregulation of cAMP/PKA pathway. Specifically, the entire repertoire of targets, many of which orchestrate processes essential for neuronal differentiation, gene reprogramming, and memory and learning, shows a trend towards hyperactivation in RBM12 depleted neurons.
    • Over 100 genes are induced in response to receptor stimulation only in the knockdown (e.g. ARC and BDNF, with crucial roles in synaptic function, plasticity, and learning.
    • RBM12 could act through other mechanisms, given that RBM12 knockdown neurons also affects the expression of genes involved in neuron differentiation, synapse organization, and neurogenesis.
    • A study on post-mortem brains of patients with bipolar affective disorder demonstrated elevated levels of the PKAcat subunit Cα in temporal and frontal cortices compared to matched normal brains.
    • A different report on patient-derived platelet cells found that the catalytic subunit of cAMP-dependent protein kinase was significantly upregulated in untreated depressed and manic patients with bipolar disorder compared with untreated euthymic patients with bipolar disorder and healthy subjects.
    • Mutations in the schizophrenia risk gene histone methyltransferase SET domain-containing protein 1 A (SETD1A) also led to transcriptional and signaling signatures supporting hyperactivation of the cAMP pathway through upregulation of adenylyl cyclases and downregulation of PDEs. This in turn resulted in increased dendritic branching and length and altered network activity in human iPSC-derived glutamatergic neurons.

    Beautiful follow up to their PLOS Genetics paper, and compelling pathological mechanism in combination with the recent SETD1A Cell Reports paper.

    Future work:

    • Does loss of RBM12 increase dendritic branching and length and alter network activity in human iPSC-derived glutamatergic neurons (e.g. does it phenocopy loss of SETD1A).
    • Does pharmacologically targeting the cAMP pathway rescue the phenotypes caused by loss of RBM12?
    • If RBM12 is ubiquitously expressed, why is the disease neuronal? What is the relevant GPCR/neuronal mechanism?
    • How does RBM12 affect the abundance of the transcripts encoding the GPCR/cAMP effectors?
    • Do mutations in any of these other rare risk genes converge on GPCR? (transcriptional regulation (SP4, RB1CC1), splicing (SRRM2). signaling (AKAP11). ion transport (CACNA1G, GRIN2A, GRIA3), neuronal migration and growth (TRIO), nuclear transport (XPO7). ubiquitin ligation (CUL1, HERC1))
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    Reply to the reviewers

    The authors do not wish to provide a response at this time

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors presented a comprehensive analysis of the effects of RBM12 on cAMP signaling and cAMP-induced transcription. All data point to hyperactivity in the absence of RBM12, suggesting that RBM12 negatively regulates cAMP signaling, particularly transcriptional response to CAMP in the nucleus. The authors found that increased expression of two adenylyl cyclase isoforms and reduced expression of PKA regulatory subunit and some isoforms of cAMP-destroying PDE is the molecular basis of excessive cAMP signaling in the absence of RBM12. The authors showed that two disease-associated RBM12 mutants are loss-of-function, as, in contrast to WT RBM12, they fail to normalize cAMP signaling and transcriptional response. The authors should be commended for confirming their findings in iPSC-derived neurons. The study is well performed and clearly described.


    Identification of earlier unappreciated mechanism regulating cAMP signaling has broad implications.

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    Referee #2

    Evidence, reproducibility and clarity


    The authors have previously carried out a CRISPR screen of b2-AR regulators of transcriptional responses. In this manuscript, the authors proceed to characterize one of the top candidates from this screen, RNA-binding motif 12 (RBM12). They perform numerous studies in HEK293 cells and iPSC-derived neurons to show that loss of RBM12 leads to a hyperactive response of ligand-induced b2AR transcription, cAMP responses and PKA activity. This increase in transcriptional responses is independent in changes of b2AR receptor expression or internalization and can be observed upon activation of other Gs-coupled GPCRs, namely ligands for adenosine A1/2R and dopamine D1R. The hyperactive transcriptional responses can also be mimicked in wild-type cells with forskolin, isoproterenol plus phosphodiesterase inhibitor, or use of cAMP analog 8-CPT-cAMP. The authors also show that variants of RPM12, that lead to familial psychosis, show hyperactive responses to isoproterenol and cannot rescue loss of wild-type RPM12. Finally, transcriptomics of loss of RPM12 in iPSC-derived neurons show altered transcriptional profiles upon stimulation with b2AR agonists.

    Major comments

    Overall this is a comprehensive study of the effects of RBM12 on cAMP-dependent transcriptional responses. The study is highly rigorous and the authors generate several novel findings, supplying a mechanism for disease-altering variants for RBM12. There are a few issues that distract somewhat as detailed below.

    1. The authors are highly focused on the GPCR responses; thus, they fail to discuss the fact that supplemental figures 1D-F show that the effects of RBM12 lie downstream of the receptor and are independent of b2AR. Stimulation with forskolin shows prominent enhancement of cAMP accumulation, pointing to enhancement of adenylyl cyclase and/or decreases in PDE activity. This effect should be quantitated. The is consistent with the fact that stimulation with multiple Gs-coupled receptors show similar enhancements. Given that, much of Fig 2, particularly 2E should be moved to supplemental. The order of figures may need to be re-examined.
      • 1b. The cAMP responses in Supplemental Fig 3 should also be quantitated with statistics.
    2. The use of 8-CPT-cAMP is not appropriate as a pure cAMP analog. It not only activates PKG, but it can also increase cGMP due to inhibition of phosphodiesterases that breakdown cGMP (PDE5).
    3. It is not clear why the authors are overexpressing the b2AR in the iPSC-derived neurons. The application of isoproterenol under conditions of overexpressed receptor is likely similar to stimulating the cell with forskolin or any agonist of an overexpressed Gs-coupled receptor. Thus it appears to be a stretch to call these "b2AR Targets". Moreover, although it is true that loss of PDE activity and/or RII subunits may contribute to loss of compartmentalization of signaling, overexpression of the GPCR could also lead to loss of compartmentalization. This must be discussed.
      • 3b. The actual list of genes in Table 3 should be shown (not just GO terms).
      • 3c. The fact that PDE1C was decreased could point to more than just cAMP-induced transcriptional changes. This is a dual PDE and its decrease may also increase cGMP.

    Minor comments:

    Missing "ISO" labels for Fig S2 C, D Need better labeling of bars for Fig 7B


    This is a very rigorous and detailed study to characterize a novel regulator of cAMP signaling systems. Although the data do not support, RBM12 as a specific regulator of beta2AR signaling, it is nevertheless an important regulator of general cAMP signaling and could potentially have effects on cGMP signaling. The study has clinical value, as RPM12 variants drive familial psychosis but this study will also appeal to basic scientists.

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    Referee #1

    Evidence, reproducibility and clarity

    RBM12 is an RNA-binding protein predominantly localized to the nucleus. Mutations in RBM12 have been linked to heritable psychosis and neurodevelopmental defects. The gene appeared in a previously published CRISPRi screen for potential regulators of cAMP signaling. The authors confirm that loss of RPM12 leads to increased basal and induced cAMP, activation of cAMP-dependent protein kinase, and induction of cAMP-CREB induced gene transcription. Similar effects were seen following direct activation of adenylyl cyclase and overexpression of the kinase catalytic subunit. The figures are clearly presented, well controlled, and show significant differences that largely support the central conclusion that RBM12 regulates cAMP. The authors have taken on an extremely challenging problem. The paper is very well written.


    The significance of the findings are limited for a number of reasons, which the authors acknowledge as summarized under major comments. We know from prior CRISPRi work that RBM12 regulates cAMP signaling (ref. 8). The findings are publishable, but the advance is modest and the potential target audience is specialized.

    Major comments.

    First, the authors are not able to mechanistically link RBM12 to any particular component of the cAMP pathway. Without a mechanistic link, direct or otherwise, to proteins to make or degrade cAMP, the findings are descriptive.

    OPTIONAL: Does RBM12 bind to and regulate a subset of mRNAs or proteins that are part of the pathway?

    Second, while the link to psychosis and neurodevelopmental defects figures very prominently in the title and text, the link to psychosis is not supported by the approach and most of the text should be removed. The experiments performed here don't come close to recapitulating the physiological setting. Is there ever a situation where individuals don't express any RBM12 (patients with the variants will be heterozygous)?

    OPTIONAL: What happens in a mouse model?