Review coordinated by Life Science Editors Foundation Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. Potential Conflicts of Interest: None.

Punchline:The liver and lung microenvironments influence various phenotypes of metastasized triple-negative breast cancer (TNBC) cells in different ways. Liver microenvironment accelerates disease progression and negatively impacts patient outcomes. Interesting paper and seems well done, identifies specific players in the different environment – consistent with the concept that the niche affects the phenotype of metastatic outgrowths.

Why is this an important problem? Metastasis is the primary cause of cancer-related deaths. Understanding how different organ environments influence the behavior of metastatic cancer cells is crucial for developing effective treatments for Stage IV disease. This research specifically focuses on TNBC, an aggressive breast cancer subtype, and its behavior in lung and liver metastatic sites.

What did we already know? * • Breast cancer frequently metastasizes to bone, liver, lungs, and brain. * • TNBC, a particularly aggressive subtype, primarily spreads to visceral organs, mainly the lungs and liver. * • The presence of bone metastases is associated with a less aggressive disease course compared to other sites. * • There are conflicting data on the aggressiveness of disease in the context of lung and liver metastases. * Results: * • Patients with liver metastases, as their first metastatic event, showed significantly shorter overall survival and time to the next metastatic event compared to those with lung metastases. Focused on a subset of patients with "mono-metastasis", meaning that a single metastasis was present in either the lung (12/318 patients with metastases) or liver (10/318 patients with metastases) at the initial diagnosis of metastasis. Kaplan-Meier plots of the progression and survival differences between patients with lung versus liver mono-metastases support the idea that liver metastases are associated with more aggressive disease progression compared to lung metastases in breast cancer patients. * * • Barcode labeling and metastasis clonality assessment in a mouse model of TNBC: mouse TNBC cells (MVT1) were tagged with unique genetic barcodes before being injected into the mammary fat pad of mice. The barcode composition of cells in the circulation (circulating tumor cells) and from each site (liver, lung) was analyzed. A greater number of unique barcodes were found in the lung metastases compared to the liver. Principal component analysis revealed that lung metastases were a distinct population compared to liver metastases and circulating tumor cells. * • After mammary fat pad injection & metastasis, mouse MVT1 or 4T1 cancer cells were isolated from either the lungs or liver, injected into the tail veins of healthy mice. Liver-resident TNBC cells have an enhanced ability to establish secondary metastases at lung and liver when compared to lung-resident cells. * * • MVT1 mouse TNBC cells expressed GFP and a soluble mCherry protein that is taken up by neighboring niche cells. Identified and analyzed both tumor and niche cell populations. * • Single-cell RNA sequencing of cancer cells: MTV1 metastatic TNBC cells have distinct molecular profiles depending on where they reside: energy generation pathways in liver-resident cells (might contribute to their increased metastatic activity) and stress and detoxification pathways in lung-resident cells (suggests a less favorable environment). * • Single-cell RNA sequencing of niche cells: Endothelial cells in the liver metastatic niche, unlike the primary tumor niche and lung niche. The unique abundance of endothelial cells in the liver niche suggests their potential involvement in shaping the metastatic behavior of liver-resident TNBC cells. Specific ligand-receptor pairs suggest a distinct communication network between cancer cells and their niche in the liver and lungs, whereby the liver niche is enriched in endothelial cells secreting Bmp2 and Bmp6 cytokines, while the lung niche is enriched in macrophages secreting Grn and Ssp1. * • Specific cytokine-receptor interactions between cancer cells and their niche were identified, with BMP2/6 secreted by liver endothelial cells and Granulin secreted by lung macrophages. * * • In vivo CRISPR-Cas9 screen used to investigate the roles of the identified cytokine-receptor pairs in metastasis formation. MTV1 TNBC cells were engineered with loss-of-function of the receptors for BMP2/6 (BMPR2, BMPR1A, and ACVR1) implicated in liver metastasis and Granulin (TNFRSF1A/B) implicated in lung metastasis. The engineered cells were injected into the tail vein and allowed to metastasize, isolated from lung and liver, and the abundance of different gene knockouts was analyzed. Additionally, they were reinjected into the tail vein to detect secondary metastasis. * * • Using this approach the organ of primary metastasis influenced the secondary metastasis (lung went to lung, liver to liver) - in contrast to when the experiment was done with WT cells injected into the mammary fat pad and isolated from liver and lung (when liver-derived TNBC showed higher secondary metastasis by tail vein to lung and liver, Fig 1I). This disconnect is a bit confusing. * * • There was no difference between the knockouts. This lack of organ specific knockouts between lung- and liver-resident cells could be due to the pleiotropic role of many of these receptors and the existence of interactions with additional cytokines. Notably, the expression of these receptors in cancer cells at the RNA level was similar in liver and lung metastases. * • Kaplan-Meier curve compares the survival of mice re-injected with either lung- or liver-resident cancer cells. Liver metastases were associated with decreased survival when compared to lung metastasis. Consistent with liver metastases associated with more aggressive disease progression compared to lung metastases. * • To investigate the effect of the niche on TNBC cells, TNBC cells were treated with either BMP2 or Granulin in vitro before being injected into the tail vein. This simulates the effects of the liver and lung microenvironment, respectively. BMP2 pre-treatment of TNBC cells enhanced metastasis formation in the lung, whereas Granulin treatment suppressed it. Supports the model that the liver niche boosts metastasis through BMP2, while the lung niche inhibits it through Granulin. This is a bit confusing with the apparent tropism of liver to liver in figure 5 but more consistent with figure 1.

What's new? Sheds light on how the liver and lung microenvironments (endothelial vs macrophage, respectively) distinctly influence the behavior of TNBC cells and why liver metastasis is associated with poor survival. Offers potential therapeutic targets for liver vs lung metastases. The discovery of the contrasting roles of BMP2 and Granulin, and their cellular sources within the respective metastatic niches, is interesting.

Potential impact * • Treatment strategies for TNBC patients with liver or lung metastases could potentially be tailored based on the identified niche-specific vulnerabilities. * • Targeting BMP2 signaling in liver metastases could potentially reduce secondary spread. * • Stimulating Granulin activity could offer a new approach to inhibiting TNBC metastasis.

Limitations: * • The study primarily focused on TNBC, and further research is needed to determine if these findings apply to other cancer types metastasizing to the liver and lungs. * • I would have liked to see preclinical models with xenografts. Therapeutic potential not shown. * • The CRISPR-Cas9 screen did not identify organ-specific knockouts, likely due to the pleiotropic roles of the targeted receptors and the complex interplay of various cytokines within the niche.

Future work: * • Investigate the applicability of these findings to other cancer types with similar metastatic patterns. * • Further explore the complex interplay of cytokines and signaling pathways within the liver and lung metastatic niches. * • Develop therapeutic strategies to target BMP2 signaling in liver metastases and stimulate Granulin activity in lung metastases. * • Validate these findings in a larger cohort of patients to determine their clinical relevance.

Review coordinated by Life Science Editors Foundation

Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. *Assisted substantially by NotebookLM.

Potential Conflicts of Interest: Angela thinks Olivia Rissland is everything a scientist should be.

What is an N-degron? N-degrons are short amino acid sequences located at the N-terminus of a protein that signal for the protein's degradation. This process is an essential part of protein quality control and regulation within cells. N-degrons are recognized by specific E3 ubiquitin ligases, also known as N-recognins, which help target the protein for degradation by the ubiquitin-proteasome system.

How was this new Arg/N-degron pathway discovered? The authors were initially studying how N-terminal sequences affect gene expression using a reporter gene assay. They found that a specific tripeptide motif (KIH) inserted at the N-terminus of a reporter protein led to a dramatic decrease in protein expression. Further investigation revealed that this decrease was due to rapid protein degradation, indicating the presence of a novel N-degron.

What are the key features of this new N-degron pathway? This newly discovered N-degron pathway targets proteins with a lysine (K) or arginine (R) residue at the third position (position 3) from the N-terminus. Importantly, this pathway requires: * • Methionine Removal: The initiator methionine (M) at position 1 must be removed by the enzyme methionine aminopeptidase 2 (MetAP2) for the degron to be active. * • UBR4 Recognition: The E3 ligase UBR4, but not UBR1 or UBR2, recognizes this specific degron and initiates the degradation process.

Why is the identity of the second amino acid important? The second amino acid plays a crucial role in determining whether MetAP2 can cleave the initiator methionine. This study found that the degron is only active when the second amino acid is threonine (T) or valine (V). These amino acids allow MetAP2 to remove the methionine, exposing the lysine or arginine at position 3 for recognition by UBR4. In contrast, if the second amino acid is alanine (A) or serine (S), MetAP1 removes the methionine. The researchers hypothesize that these N-termini are then acetylated, preventing UBR4 recognition.

Is there evidence that this pathway affects endogenous proteins? Yes, analysis of previously published data and additional experiments by the researchers suggest that this MetAP2-UBR4 pathway is not limited to artificial reporter systems. They found that endogenous proteins with MTK or MVK N-termini were less stable than those with other amino acids at position.

Does UBR4 work alone in this pathway? UBR4 appears to function as part of a complex with the protein KCMF1 to degrade proteins containing this new degron. Experiments showed that disrupting the UBR4-KCMF1 complex stabilized the degradation of reporter proteins containing the KIH degron.

What is the broader significance of this discovery? The identification of this new Arg/N-degron pathway expands our understanding of the N-end rule, a fundamental mechanism for protein degradation in cells. It highlights the complexity of this system and reveals how the interplay between different enzymes like MetAP2 and E3 ligases like UBR4 can fine-tune protein stability. Additionally, it suggests that there may be other undiscovered N-degron pathways that remain to be characterized.

What questions still need to be answered about this new pathway? This study raises several new questions, including: * • Substrate Specificity: What are the precise rules governing UBR4 recognition of position 3 lysine and arginine degrons? Do other amino acids in the protein sequence affect degron recognition? * • Physiological Roles: What are the specific cellular processes and pathways regulated by this MetAP2-UBR4 N-degron pathway? * • Evolutionary Conservation: Is this pathway conserved in other organisms, or is it unique to mammals? * • Therapeutic Potential: Could this pathway be targeted for therapeutic purposes? For example, could stabilizing proteins involved in disease by manipulating this pathway be beneficial?

What was not known: * • Whether a lysine or arginine residue at position 3 of a protein could act as an N-degron. * • Whether MetAP2 could play a role in initiating N-degron-mediated degradation.

What this preprint reveals: * • A new family of N-degrons: The study identified a new class of N-degrons characterized by a lysine or arginine residue at position 3, following a methionine at position 1 and a MetAP2-cleavable residue (threonine or valine) at position * • MetAP2-dependent initiation of the Arg/N-degron pathway: The study found that MetAP2-mediated removal of the initiator methionine is essential for the recognition and degradation of these position 3 lysine/arginine degrons. This is the first demonstration of MetAP2's involvement in this pathway * • UBR4 as the primary E3 ligase: UBR4, rather than UBR1 or UBR2, was identified as the primary E3 ligase responsible for recognizing and targeting proteins with the newly identified position 3 degrons for degradation. * • Role of downstream residues: The study showed that amino acid residues downstream of the position 3 lysine/arginine can influence both methionine cleavage by MetAP2 and recognition by UBR4, highlighting the complexity of the N-degron pathway. * • Endogenous protein regulation: The study provided evidence suggesting that this MetAP2-dependent, UBR4-mediated Arg/N-degron pathway regulates the stability of endogenous proteins, highlighting its broader biological significance.

Ang's take- somewhat specialized and 'ectopic' but important, thorough, and unambiguous. Satisfying. Very likely to be physiologically relevant even though most of the assays were done with reporters. Regardless, showing that this rule 'is' true is useful for technological applications.

Review coordinated by Life Science Editors Foundation

Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. *Assisted by NotebookLM.

Potential Conflicts of Interest: None

Under review at Nature Portfolio

Punchline: Neurons under stress can locally synthesize Heat Shock Proteins (HSPs) in dendrites by increasing the transport of their mRNAs from the soma.

Why is this interesting? This is a previously unknown mechanism for locally synthesizing HSPs in neuronal dendrites in response to stress. It could shed light on therapeutic strategies for neurodegenerative diseases, which are characterized by a loss of proteostasis.

Background:

• • Maintaining proteostasis is difficult for neurons because of their complex polarized morphology and the need for constant remodeling of the synaptic proteome.
• • Local Translation in Neurons: The concept of local translation, particularly within neuronal dendrites, was already well-established. mRNA localization and local translation are fundamental processes in neurons, allowing for spatial and temporal control of protein synthesis. This is particularly crucial in dendrites, which are distant from the soma and require localized protein synthesis for synaptic plasticity and other functions.
• • HSPs and Proteostasis: The importance of heat shock proteins (HSPs) in maintaining cellular proteostasis was also well-understood. HSPs act as chaperones, assisting in the proper folding of proteins and preventing the formation of harmful aggregates.
• • RNA-Binding Proteins and mRNA Localization: RNA-binding proteins (RBPs) play a critical role in regulating mRNA localization and translation. These proteins often recognize mRNAs and direct their transport to specific subcellular locations.

Results: * • When hippocampal and spinal cord motor neurons are stressed, they increase the transport of HSP mRNAs to the dendrites. * • Used a variety of techniques to stress the neurons, including inhibiting the proteasome, hypoxia, and exposure to amyloid-beta peptides. * • All of these stresses led to an increase in the levels of HSP mRNAs in the dendrites. * • The increase in HSP mRNA levels in the dendrites was accompanied by an increase in the levels of HSP proteins in the dendrites. * • This suggests that the HSP mRNAs are being translated into proteins in the dendrites. * • Transport of HSP mRNAs to the dendrites was dependent on the microtubule motor protein dynein. * • Two RNA-binding proteins, FUS and HNRNPA2B1, regulate the transport of HSP mRNAs to dendrites. * • Depletion of FUS or expression of the ALS-associated HNRNPA2B1 D290V mutation impaired the dendritic localization of HSP mRNAs in mouse and human motor neurons.

Discussion: * • Stress-Responsive HSP mRNA Transport and Translation in Dendrites: While previous studies had identified local translation of some proteins in dendrites and recognized the role of HSPs in neurons, this paper specifically focuses on the regulated transport and localized translation of HSP mRNAs in dendrites as a key mechanism for responding to proteotoxic stress. This adds a new layer of understanding to neuronal stress responses. * • Identification of FUS and HNRNPA2B1 as Key Regulators: The study goes a step further by identifying and characterizing the specific roles of RNA-binding proteins FUS and HNRNPA2B1 in regulating HSP mRNA transport. This mechanistic insight into how HSP mRNA localization is controlled enhances our understanding of how neurons fine-tune proteostasis in a spatially defined manner. * • Linking HSP mRNA Localization to ALS: The study makes a significant connection between the dysregulation of HSP mRNA localization and amyotrophic lateral sclerosis (ALS). By demonstrating that an ALS-associated mutation in HNRNPA2B1 (D290V) impairs HSPA8 mRNA localization and increases neuronal vulnerability, the study provides a potential molecular mechanism for this devastating neurodegenerative disease. This link between impaired local translation, proteostasis, and ALS opens up new avenues for research and potential therapeutic interventions.

Limitations: • Experiments conducted in cultured neurons.

Future work: * • Investigate the role of this mechanism in vivo. * • Determine whether this mechanism is impaired in other neurodegenerative diseases. * • Inform therapeutic strategies that can target this mechanism to treat or prevent neurodegenerative diseases.

Selected Reading 1. Bourke, Ashley M. et al. De-centralizing the Central Dogma: mRNA translation in space and time Molecular Cell, Volume 83, Issue 3, 452 – 468 (2023) 2. Davidson, Alexander et al. Localized Translation of gurken/TGF-α mRNA during Axis Specification Is Controlled by Access to Orb/CPEB on Processing Bodies Cell Reports, Volume 14, Issue 10, 2451 – 2462 (2016) 3. Gehrke, Stephan et al. PINK1 and Parkin Control Localized Translation of Respiratory Chain Component mRNAs on Mitochondria Outer Membrane Cell Metabolism, Volume 21, Issue 1, 95 – 108 (2015) 4. Hacisuleyman, E., Hale, C.R., Noble, N. et al. Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding. Nat Neurosci 27, 822–835 (2024). 5. Höpfler, Markus et al. Control of mRNA fate by its encoded nascent polypeptide Molecular Cell, Volume 83, Issue 16, 2840 – 2855 (2023) 6. Park, Sungjin et al. The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation Developmental Cell, Volume 58, Issue 19, 1917 - 1932.e6 (2023) 7. Lautier, Ophélie et al. Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis Molecular Cell, Volume 81, Issue 11, 2417 - 2427.e5 (2021) 8. Ramat, A., Haidar, A., Garret, C. et al. Spatial organization of translation and translational repression in two phases of germ granules. Nat Commun 15, 8020 (2024).

Review coordinated by Life Science Editors Foundation

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

Potential Conflicts of Interest: None

Punch line: Activation of the yeast AMP-activated protein kinase (AMPK) negatively regulates MAGIC, inhibits the import of misfolded proteins into mitochondria & promotes mitochondrial biogenesis and fitness.

Why is this interesting? Maybe all those healthy things like caloric restriction, intermittent fasting, exercise etc that activate AMPK & extend lifespan do so by inhibiting MAGIC & preventing mitochondrial damage from misfolded proteins.

Background: Metabolic imbalance & loss of proteostasis are interconnected hallmarks of aging and age-related diseases. A mitochondria-mediated proteostasis mechanism called MAGIC (mitochondria as guardian in cytosol) concentrates cytosolic misfolded protein at the surface of mitochondria, where they are disaggregated by molecular chaperones, and then imported for degradation by mitochondrial proteases. Inhibition of this pathway prolongs protein aggregation in cytosol after proteotoxic stress, but excessive misfolded proteins in mitochondria can lead to mitochondrial damage.

Results: • Genetic screen for MAGIC regulators uncovered 145 genes. Loss of Snf1 (AMPK homolog) led to increased mitochondrial import even without proteotoxic stress. In contrast indirect, constitutive activation of Snf1 (e.g. low glucose) prevented the import of misfolded proteins in mitochondria.

• The data suggest that the reduced accumulation of misfolded proteins in mitochondria of Snf1-active cells is not due to enhanced intramitochondrial degradation nor to reduced levels of the misfolded protein, but rather due to blocked mitochondrial import.

• Deletion of HAP4 counteracted Snf1 activation and overexpression of Hap4 alone recapitulated Snf1 activation. The Hap2/3/4/5 complex activates the expression of nuclear encoded mitochondrial proteins. Their data suggest that high expression of mitochondrial preproteins due to an elevated Snf1-Hap4 axis compete with misfolded proteins for mitochondrial import.

• Proteotoxic stress led to a reduced growth rate & reduced mitochondrial fitness in high glucose medium but not under glucose limitation. The data suggest that low glucose, activation of Snf1 & prevention of misfolded protein import into mitochondria prevent the growth defect.

• Many neurodegenerative disease-associated aggregation-prone proteins (α-synuclein, FUSP525L, TDP-43, amyloid beta, C9ORF72-associated poly(GR) dipeptide) are detected in mitochondria of human patients or disease models and impair mitochondrial functions. Their data suggest that the import of α-synuclein & associated reduction in mitochondrial fitness can be counteracted by indirect AMPK/Snf1 activation (i.e. glucose limitation).

• Show data in yeast & human cells.

Discussion: This paper revealed an unexpected link between cellular metabolism and proteostasis through MAGIC/mitochondria.

• Snf1/AMPK is a key regulator of MAGIC & of misfolded protein import into mitochondria.

• Snf1/AMPK balances the mitochondrial metabolic and proteostatic functions in response to glucose availability and protects mitochondrial fitness under proteotoxic stress.

• The authors speculate that in high glucose, cells rely on glycolysis for ATP production and mitochondria ‘moonlighting’ in cellular proteostasis through MAGIC, but when glucose is limited and cells rely on oxidative phosphorylation for ATP generation, AMPK is activated and shuts down MAGIC, prioritizing the import of essential mitochondrial preproteins to ensure mitochondrial fitness and energy production.

• Acknowledge limitations: Snf1/Hap4 activation elevates the expression of hundreds of mitochondrial preproteins, not clear whether specific preproteins or cytosolic factors directly involved in inhibiting mitochondrial import, & that more details on mechanisms will be of interest.

• Caloric restriction & AMPK activation might contribute to lifespan extension by inhibiting MAGIC. In human, AMPK activity is elevated during health-benefitting activities such as exercise. Their data suggest that elevating AMPK activity may be beneficial in alleviating proteotoxicity associated with degenerative diseases - but hyperactivated AMPK has also been reported in several neurodegenerative diseases with proteostasis decline (Ang wonders- maybe AMPK is overwhelmed?).

THIS IS A GORGEOUS PAPER!

Future work - I can't wait to see the characterization of the ribosome biogenesis genes that they also pulled out as MAGIC regulators. Anticipating a translation, misfolded protein, mitochondria, aging axis :)

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.

Background: * 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.

Results: * 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.<br /> * 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.

Discussion: * 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))

Review coordinated by Life Science Editors Foundation

Reviewed by: Dr. Angela Andersen

Potential Conflicts of Interest: None

Background: * mRNAs in polarized cells often have a distinct spatial localization patterns that enable localized protein production * In non-polarized cells, mRNAs encoding membrane and secretory proteins are predominantly translated on the endoplasmic reticulum (ER), some mRNAs are enriched on the mitochondrial surface, some mRNAs are bound to the RNA-binding protein (RBP) TIS11B at the surface of the rough ER in "TIS granules". * The translation of specific mRNAs in TIS granules allows assembly of protein complexes that cannot be established when the mRNAs are translated on the ER but outside of TIS granules (physiological relevance). * The canonical rough ER (CRER) is distinct from the TIS granule ER (TGER), and both are distinct from the cytosol.

Questions: * Do mRNAs that encode non-membrane proteins differentially localize to the ER or the cytosol? (in steady state) * Does the amount of protein synthesis differ depending on the subcytoplasmic location of an mRNA?

Summary: * A third of mRNAs that encode non-membrane proteins have a biased localization to TGER or CRER, indicating that the ER membrane is a general site of translation for both membrane and non-membrane proteins. * 52% of mRNAs that encode non-membrane proteins have a biased mRNA transcript localization pattern towards a single cytoplasmic compartment. the TGER, CRER or cytosol. * The localization at the TGER or CRER was largely controlled by a combinatorial code of AU-RBPs at the 3'UTR. TIS11B promotes mRNA localization to TGER and TIA1/L1 to CRER. * LARP4B bound to the 3'UTR promotes cytosolic localization. * The location of translation has an independent effect on protein levels independent of the RBPs/3'UTR: redirecting cytosolic mRNAs to the rough ER membrane increased their steady-state protein levels by two-fold, indicating that the ER environment promotes protein expression. * Compartment-enriched mRNAs differed in their mRNA production and degradation rates, as well as functional classes and levels of their encoded proteins. Therefore the cytoplasm is partitioned into different functional and regulatory compartments that are not enclosed by membranes. * low-abundance proteins are translated in the TGER region. mRNAs encoding zinc finger proteins and transcription factors were substantially enriched at the TGER. These gene classes are usually expressed at lower levels than others.. This localization may regulate protein complex assembly (membrane proteins that are translated in the TGER domain establish protein complexes that cannot be formed when the proteins are translated on the CRER). The TGER may ensure that low-abundance mRNAs are effectively translated into low-abundance proteins. * mRNAs that are the most stable and encode the most highly expressed proteins are enriched on the CRER and include helicases, cytoskeleton-bound proteins, and chromatin regulators, overturning the idea that most non-membrane protein-encoding mRNAs are translated in the cytosol. * mRNAs overrepresented in the cytosol had the highest production and degradation rates and were enriched in proteins involved in mRNA processing and translation factors, whose abundance levels require tight control.

Advance: Evidence for functional compartmentalization of non-membrane mRNA protein expression in the cytosol vs ER. In steady state, general localization of mRNAs to the ER promotes high protein levels.

Significance: Engineered 3'UTR sequences could potentially boost protein expression by localizing mRNAs to the ER in experimental settings, for vaccines etc.

Remaining questions/points: * How does the rough ER stimulate protein expression? * Does the mRNA localization affect complex formation and/or function of non-membrane proteins? * Does this occur in cells other than HEK293T? * Is this regulated?