linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells
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eLife assessment
This paper will be of interest to scientists involved in understanding the function of long non-coding RNAs. The authors found two genes previously reported as lincRNAs in early studies encode micropeptides in zebrafish. Zebrafish mutants lacking these micro-peptides show altered gene regulatory networks that preferentially affect oligodendrocytes and cerebellar cells in the embryonic brain. The data presented in the study are solid and present convincing additional evidence for the versatile functions of micro-peptides.
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Abstract
Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs ( linc-mipep, also called lnc-rps25, and linc-wrb ) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes – evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.
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Author Response
Reviewer #2 (Public Review):
The two new micropeptides are well characterized in the manuscript and appear to be functionally important with some chromatin-level consequences of their loss (which can be either direct or indirect), but the finding that lincRNA sequences encode micropeptides is not novel, and the two described in the paper appear to be zebrafish-specific and their function was tested only in zebrafish, which limits the interest in these genes. The use of ribosome profile data along behavioral screening to identify micropeptides is interesting and important, but the scope of the screen, the candidates selected for testing, etc. are not clear enough as presented. The ChIP-seq analysis of the new proteins is very interesting but is not described in any detail. Overall, the experimental part is well …
Author Response
Reviewer #2 (Public Review):
The two new micropeptides are well characterized in the manuscript and appear to be functionally important with some chromatin-level consequences of their loss (which can be either direct or indirect), but the finding that lincRNA sequences encode micropeptides is not novel, and the two described in the paper appear to be zebrafish-specific and their function was tested only in zebrafish, which limits the interest in these genes. The use of ribosome profile data along behavioral screening to identify micropeptides is interesting and important, but the scope of the screen, the candidates selected for testing, etc. are not clear enough as presented. The ChIP-seq analysis of the new proteins is very interesting but is not described in any detail. Overall, the experimental part is well designed and the phenotypes reported by the authors appear to be strong and convincing, but the mechanistic understanding of what the two new proteins do and how, and the general interest in the results given the current scope of understanding of micropeptide is limited.
We apologize for the misunderstanding that these genes are zebrafish-specific. In this revision, we have clarified throughout the text and with additional data that these genes are not zebrafish-specific, but that linc-mipep and linc-wrb are homologous to human Hmgn1.
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eLife assessment
This paper will be of interest to scientists involved in understanding the function of long non-coding RNAs. The authors found two genes previously reported as lincRNAs in early studies encode micropeptides in zebrafish. Zebrafish mutants lacking these micro-peptides show altered gene regulatory networks that preferentially affect oligodendrocytes and cerebellar cells in the embryonic brain. The data presented in the study are solid and present convincing additional evidence for the versatile functions of micro-peptides.
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Reviewer #1 (Public Review):
Tornini et al. investigate the function of long non-coding RNAs in vivo. In the manuscript, the authors show that two of these molecules linc-mipep and linc-wrb encode for a micropeptide that regulates zebrafish behavior. In the absence of this peptide, zebrafish larvae show dysregulation of NMDA receptor and glucocorticoid receptor-mediated signaling and immediate early gene induction. Given the homology of linc-mipep and linc-wrb encoded peptides with homology to chromosome binding and chromatin unwinding domain of HMGN1 the authors explore the altered chromatin accessibility in the mutant animals. This analysis revealed a broad dysregulation of 3D chromatin structure with some enrichment at loci regulating the expression of immediate early response genes. Finally, single cell analysis revealed that …
Reviewer #1 (Public Review):
Tornini et al. investigate the function of long non-coding RNAs in vivo. In the manuscript, the authors show that two of these molecules linc-mipep and linc-wrb encode for a micropeptide that regulates zebrafish behavior. In the absence of this peptide, zebrafish larvae show dysregulation of NMDA receptor and glucocorticoid receptor-mediated signaling and immediate early gene induction. Given the homology of linc-mipep and linc-wrb encoded peptides with homology to chromosome binding and chromatin unwinding domain of HMGN1 the authors explore the altered chromatin accessibility in the mutant animals. This analysis revealed a broad dysregulation of 3D chromatin structure with some enrichment at loci regulating the expression of immediate early response genes. Finally, single cell analysis revealed that oligodendrocyte progenitor cells and cerebellar granule cells are more affected in the mutants.
This work represents a technical tour-de-force with extensive genomics data to characterize the molecular phenotype of linc-mipep and linc-wrb loss of function. This data show interesting findings in part consistent with the behavioral phenotype observed.
The manuscript provides compelling evidence that micropeptides encoded by what were previously identified as long non-coding RNAs have a precise biological function.
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Reviewer #2 (Public Review):
The two new micropeptides are well characterized in the manuscript and appear to be functionally important with some chromatin-level consequences of their loss (which can be either direct or indirect), but the finding that lincRNA sequences encode micropeptides is not novel, and the two described in the paper appear to be zebrafish-specific and their function was tested only in zebrafish, which limits the interest in these genes. The use of ribosome profile data along behavioral screening to identify micropeptides is interesting and important, but the scope of the screen, the candidates selected for testing, etc. are not clear enough as presented. The ChIP-seq analysis of the new proteins is very interesting but is not described in any detail. Overall, the experimental part is well designed and the …
Reviewer #2 (Public Review):
The two new micropeptides are well characterized in the manuscript and appear to be functionally important with some chromatin-level consequences of their loss (which can be either direct or indirect), but the finding that lincRNA sequences encode micropeptides is not novel, and the two described in the paper appear to be zebrafish-specific and their function was tested only in zebrafish, which limits the interest in these genes. The use of ribosome profile data along behavioral screening to identify micropeptides is interesting and important, but the scope of the screen, the candidates selected for testing, etc. are not clear enough as presented. The ChIP-seq analysis of the new proteins is very interesting but is not described in any detail. Overall, the experimental part is well designed and the phenotypes reported by the authors appear to be strong and convincing, but the mechanistic understanding of what the two new proteins do and how, and the general interest in the results given the current scope of understanding of micropeptide is limited.
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Reviewer #3 (Public Review):
The study aimed at the identification of functional micro-peptides encoded by transcripts previously annotated as long noncoding RNAs (lncRNAs). The authors pre-selected 10 candidates out of the ~500 zebrafish lncRNA data set based on their engagement with the ribosome (by ribosome profiling data) and their expression in the embryonic brain. By performing an F0 CRISPR/Cas9 screen coupled with embryonic behavioral assays, two transcripts encoding sequence-related micro-peptides were identified. Using a set of stable mutant alleles, the authors showed that mutations specifically affecting the open reading frame (ORF) of the putative micro-peptides cause changes in embryonic behavior when compared to wild-type embryos or embryos with mutations in the non-coding regions of the tested transcripts. The locomotor …
Reviewer #3 (Public Review):
The study aimed at the identification of functional micro-peptides encoded by transcripts previously annotated as long noncoding RNAs (lncRNAs). The authors pre-selected 10 candidates out of the ~500 zebrafish lncRNA data set based on their engagement with the ribosome (by ribosome profiling data) and their expression in the embryonic brain. By performing an F0 CRISPR/Cas9 screen coupled with embryonic behavioral assays, two transcripts encoding sequence-related micro-peptides were identified. Using a set of stable mutant alleles, the authors showed that mutations specifically affecting the open reading frame (ORF) of the putative micro-peptides cause changes in embryonic behavior when compared to wild-type embryos or embryos with mutations in the non-coding regions of the tested transcripts. The locomotor hyperactivity phenotype was even stronger in double homozygous mutants suggesting a redundant function of both micro-peptides. The authors demonstrated that the behavioral phenotype of one of the mutants was rescued by the transgene expression of the coding sequence (CDS). Sequence analyses of both peptides revealed their conservation and homology to the human non-histone chromosomal proteins (HMGN1 proteins). The authors demonstrated that the micro-peptide mutants exhibit changes in chromatin accessibility for transcription factors modifying neural activation, dysregulation of gene expression programs, and changes in oligodendrocyte and cerebellar cell states during development.
The study presents an important discovery of two sequence-related micro-peptides with important and potentially conserved functions during development. While it is still unclear how the micro-peptides act in the cell, it is evident that they are key regulators of cellular states. Whereas the study is well done, the data presentation should be improved as several important details were omitted.
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Reviewer #4 (Public Review):
In this manuscript, Tornini and colleagues identify two previously un-characterized micropeptides encoded by linc-mipep and linc-wrb as important modulators of day-time activity in zebrafish larvae. The authors demonstrate that each single mutant shows an increase in day-time activity and that double mutants show a more pronounced effect. Of interest, ubiquitous overexpression of the ORF encoding the linc-mipep-derived peptide can rescue the day-time over-activity phenotype of linc-mipep mutant larvae, establishing that linc-mipep acts indeed as a protein and not at the level of RNA. Using a series of experimental approaches, including ATAC-Seq from double mutant brains and scRNA-Seq and scATAC-seq analyses from linc-mipep mutants as well as linc-mipep and linc-wrb CHIP analyses, the authors furthermore …
Reviewer #4 (Public Review):
In this manuscript, Tornini and colleagues identify two previously un-characterized micropeptides encoded by linc-mipep and linc-wrb as important modulators of day-time activity in zebrafish larvae. The authors demonstrate that each single mutant shows an increase in day-time activity and that double mutants show a more pronounced effect. Of interest, ubiquitous overexpression of the ORF encoding the linc-mipep-derived peptide can rescue the day-time over-activity phenotype of linc-mipep mutant larvae, establishing that linc-mipep acts indeed as a protein and not at the level of RNA. Using a series of experimental approaches, including ATAC-Seq from double mutant brains and scRNA-Seq and scATAC-seq analyses from linc-mipep mutants as well as linc-mipep and linc-wrb CHIP analyses, the authors furthermore identify differences in chromatin accessibility and gene expression in specific cell types of the larval brain in the absence of linc-mipep (and in case of globale ATAC-Seq, in the absence of both peptides). They conclude that the micropeptides regulate behavior and neuronal states by modulating chromatin accessibility, revealing functional similarities to their known vertebrate homolog HMGN1.
Overall, the key finding of this paper, namely the identification of two functional microproteins that had previously been misannotated as lincRNAs but have homology to HMGN1 both based on their sequence and function is an exciting discovery since relatively few newly predicted micropeptides have been functionally characterized to date, and because it advances our understanding of the molecular mechanisms underlying vertebrate-specific neuronal function and diversity. The F0 screen leading to the identification of 2 functional micropeptides provides a major advance to the field since so far screens in the F0 generation have not been typically done (rather germline-transmission). Thus, this work provides a major step forward in this regard. In addition, it includes a series of scRNA- and scATAC analyses that are technologically at the forefront and not easy to conduct and analyse.
The weakest part of the paper in its current form is on the one hand missing the link between the behavioral phenotype in mutants and the molecular phenotypes in the larval brain. It remains unclear how one can reconcile the broad neuronal expression (in the case of linc-mipep preferentially in Purkinje cells) and linc-wrb with the cell-specific effects. Moreover, it is not clear whether both peptides act redundantly or in parallel but distinct pathways since the rescue is only shown for the single linc-mipep mutant by linc-mipep overexpression (and no rescue is shown for linc-wrb or the double mutant). While the authors suggest throughout the manuscript that both peptides have similar functions (act redundantly), no clear data is provided for this, and the use of either single linc-mipep mutants (all single-cell analyses in the last Figure) or double linc-mipep/linc-wrb mutants (global brain ATAC-Seq analyses) for different brain analyses makes the molecular analyses inconsistent and not easy to interpret. While the overall finding(s) of the paper is really interesting, to make this paper really solid, additional controls and analyses will be needed.
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