Dilated cardiomyopathy-associated RNA Binding Motif Protein 20 regulates long pre-mRNAs in neurons
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eLife Assessment
This important study reveals a new mechanism for gene regulation in neurons by an RNA binding protein called RBM20 previously studied in the heart. The methods used are compelling, including the generation of new mouse knockout strains and leading edge sequencing methods for identification of gene regulatory mechanisms. The study shows that neuronal RBM20 governs long pre-mRNAs encoding synaptic proteins in specific neuronal cell types, but the functional consequences of this regulation remain questions for the future.
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Abstract
Precise coordination of molecular programs and neuronal growth govern the formation, maintenance, and adaptation of neuronal circuits. RNA metabolism has emerged as a key regulatory node of neural development and nervous system pathologies. To uncover cell-type-specific RNA regulators, we systematically investigated expression of RNA recognition motif-containing proteins in the mouse neocortex. Surprisingly, we found RBM20, an alternative splicing regulator associated with dilated cardiomyopathy, to be expressed in cortical parvalbumin interneurons and mitral cells of the olfactory bulb. Genome-wide mapping of RBM20 target mRNAs revealed that neuronal RBM20 binds pre-mRNAs in distal intronic regions. Loss of neuronal RBM20 has only modest impact on alternative splice isoforms but results in a significant reduction in an array of mature mRNAs in the neuronal cytoplasm. This phenotype is particularly pronounced for genes with long introns that encode synaptic proteins. We hypothesize that RBM20 ensures fidelity of pre-mRNA splicing by suppressing non-productive splicing events in long neuronal genes. This work highlights a common requirement for RBM20-dependent transcriptome regulation in cardiomyocytes and neurons and demonstrates that a major genetic risk factor of heart disease impacts neuronal gene expression.
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eLife Assessment
This important study reveals a new mechanism for gene regulation in neurons by an RNA binding protein called RBM20 previously studied in the heart. The methods used are compelling, including the generation of new mouse knockout strains and leading edge sequencing methods for identification of gene regulatory mechanisms. The study shows that neuronal RBM20 governs long pre-mRNAs encoding synaptic proteins in specific neuronal cell types, but the functional consequences of this regulation remain questions for the future.
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Reviewer #1 (Public review):
Summary:
The authors of this study set out to find RNA binding proteins in the CNS in cell-type specific sequencing data and discover that the cardiomyopathy-associated protein RBM20 is selectively expressed in olfactory bulb glutamatergic neurons and PV+ GABAergic neurons. They make an HA-tagged RBM20 allele to perform CLIP-seq to identify RBM20 binding sites and find direct targets of RBM20 in olfactory bulb glutmatergic neurons. In these neurons, RBM20 binds intronic regions. RBM20 has previously been implicated in splicing, but when they selectively knockout RBM20 in glutamatergic neurons they do not see changes in splicing, but they do see changes in RNA abundance, especially of long genes with many introns, which are enriched for synapse-associated functions. These data show that RBM20 has important …
Reviewer #1 (Public review):
Summary:
The authors of this study set out to find RNA binding proteins in the CNS in cell-type specific sequencing data and discover that the cardiomyopathy-associated protein RBM20 is selectively expressed in olfactory bulb glutamatergic neurons and PV+ GABAergic neurons. They make an HA-tagged RBM20 allele to perform CLIP-seq to identify RBM20 binding sites and find direct targets of RBM20 in olfactory bulb glutmatergic neurons. In these neurons, RBM20 binds intronic regions. RBM20 has previously been implicated in splicing, but when they selectively knockout RBM20 in glutamatergic neurons they do not see changes in splicing, but they do see changes in RNA abundance, especially of long genes with many introns, which are enriched for synapse-associated functions. These data show that RBM20 has important functions in gene regulation in neurons, which was previously unknown, and they suggest it acts through a mechanism distinct from what has been studied before in cardiomyocytes.
Strengths:
The study finds expression of the cardiomyopathy-associated RNA binding protein RBM20 in specific neurons in the brain, opening new windows into its potential functions there.
The study uses CLIP-seq to identify RBM20 binding RNAs in olfactory bulb neurons.
Conditional knockout of RBM20 in glutamatergic or PV neurons allows the authors to detect mRNA expression that is regulated by RBM20.
The data include substantial controls and quality control information to support the rigor of the findings.
Weaknesses:
The authors do not fully identify the mechanism by which RBM20 acts to regulate RNA expression in neurons, though they do provide data suggesting that neuronal RBM20 does not regulate alternate splicing in neurons, which is an interesting contrast to its proposed mechanism of function in cardiomyocytes. Discovery of the RNA regulatory functions of RBM20 in neurons is left as a question for future studies.
The study does not identify functional consequences of the RNA changes in the conditional knockout cells, so this is also a question for the future.
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Reviewer #2 (Public review):
Summary:
The group around Prof. Scheiffele has made seminal discoveries reg. alternative splicing that is reflected by a current ERC advanced grant and landmark papers in eLife (2015), Science (2016), and Nature Neuroscience (2019). Recently, the group investigated proteins that contain an RRM motif in the mouse cortex. One of them, termed RBM20, was originally thought to be muscle-specific and involved in alternative splicing in cardiomyocytes. However, upon close inspection, RBP20 is expressed in a particular set of interneurons (PV positive cells of the somatosensory cortex) in the cortex as well as in mitral cells of the olfactory bulb (OB). Importantly, they used CLIP to identify targets in the OB and heart. Next and quite importantly, they generated a knock-in mouse line with a His-biotin acceptor …
Reviewer #2 (Public review):
Summary:
The group around Prof. Scheiffele has made seminal discoveries reg. alternative splicing that is reflected by a current ERC advanced grant and landmark papers in eLife (2015), Science (2016), and Nature Neuroscience (2019). Recently, the group investigated proteins that contain an RRM motif in the mouse cortex. One of them, termed RBM20, was originally thought to be muscle-specific and involved in alternative splicing in cardiomyocytes. However, upon close inspection, RBP20 is expressed in a particular set of interneurons (PV positive cells of the somatosensory cortex) in the cortex as well as in mitral cells of the olfactory bulb (OB). Importantly, they used CLIP to identify targets in the OB and heart. Next and quite importantly, they generated a knock-in mouse line with a His-biotin acceptor peptide and a HA epitope to perform specific biochemistry. Not surprisingly, this allowed them to specifically identify transcripts with long introns, however, most of the intronic binding sites were very distant to the splice sites. Closer GO term inspection revealed that RBM20 specifically regulates synapse-related transcripts. In order to get in vivo insight into its function in the brain, the authors generated both global as well as conditional KO mice. Surprisingly, there were no significant differences in in RBM20 ΔPV interneurons, however, 409 transcripts were deregulated in in OB glutamatergic neurons. Here, CLIP sites were mostly found to be very distant from differentially expressed exons. Furthermore, loss-of-function RBM20 primarily yields loss of transcripts, whereas upregulation appears to be indirect. Together, these results strongly suggest a role of RBM20 in the inclusion of cryptic exons thereby promoting target degradation.
Strengths:
The quality of the data and the figures is high, impressive and convincing. The reported results strongly suggest a role of RBM20 in the inclusion of cryptic exons thereby promoting target degradation.
Weaknesses:
I would not use the term weakness here.
The description of the results is sometimes too dense and technical. As eLife does not have a size limit, there is no reason for the results section to be less than three pages. Especially the last paragraph of the results part (p4) does not do justice to the high importance of Fig. 5, which I consider of high importance and originality. Here are a few suggestions from a person that is not working on splicing, to improve the text part of this important manuscript.(1) Introduction: too short, include a paragraph on splicing and cryptic exons
(2) Results:
- shortly describe the phenotypes of the mice mentioned
- expand the section on Fig. 5 and cryptic exons especially
(3) Discussion: too short, expand on the possible new role of RBM20 and target degradation, possibly by adding a scheme? -
Reviewer #3 (Public review):
Summary:
The authors identified RBM20 expression in neural tissues using cell type-specific transcriptomic analysis. This discovery was further validated through in vitro and in vivo approaches, including RNA fluorescent in situ hybridization (FISH), open-source datasets, immunostaining, western blotting, and gene-edited RBM20 knockout (KO) mice. CLIP-seq and RiboTRAP data demonstrated that RBM20 regulates common targets in both neural and cardiac tissues, while also modulating tissue-specific targets. Furthermore, the study revealed that neuronal RBM20 governs long pre-mRNAs encoding synaptic proteins.
Strengths:
• Utilization of a large dataset combined with experimental evidence to identify and validate RBM20 expression in neural tissues.
• Global and tissue-specific RBM20 KO mouse models provide robust …Reviewer #3 (Public review):
Summary:
The authors identified RBM20 expression in neural tissues using cell type-specific transcriptomic analysis. This discovery was further validated through in vitro and in vivo approaches, including RNA fluorescent in situ hybridization (FISH), open-source datasets, immunostaining, western blotting, and gene-edited RBM20 knockout (KO) mice. CLIP-seq and RiboTRAP data demonstrated that RBM20 regulates common targets in both neural and cardiac tissues, while also modulating tissue-specific targets. Furthermore, the study revealed that neuronal RBM20 governs long pre-mRNAs encoding synaptic proteins.
Strengths:
• Utilization of a large dataset combined with experimental evidence to identify and validate RBM20 expression in neural tissues.
• Global and tissue-specific RBM20 KO mouse models provide robust support for RBM20 localization and expression.
• Employing heart tissue as a control highlights the unique findings in neural tissues.Weaknesses:
• Lack of physiological functional studies to explore RBM20's role in neural tissues.
• Data quality requires improvement for stronger conclusions.
• Western blot sample size should be increased for enhanced reliability. -
Author response:
We thank the reviewers for the constructive suggestions made in the Public Reviews and the Recommendations to Authors. We intend to address these comments in a revised manuscript as follows:
(1) We will revise the text according to the reviewer suggestions with regards to specific RBM20-dependent mRNAs and providing more detailed explanations in results and discussion.
(2) We will upload higher resolution images of several figures (resolution had been reduced to achieve lower file sizes) to address the comment regarding “data quality”.
(3) We will include data on eCLIP control experiments.
(4) We will add information on replication and new data for the western blot analysis.
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