Tamara Sirey

A Transcriptomic Atlas of Mouse Neocortical Layers

Summary In the mammalian cortex, neurons and glia form a patterned structure across six layers whose complex cytoarchitectonic arrangement is likely to contribute to cognition. We sequenced transcriptomes from layers 1-6b of different areas (primary and secondary) of the adult (postnatal day 56) mouse somatosensory cortex to understand the transcriptional levels and functional repertoires of coding and noncoding loci for cells constituting these layers. A total of 5,835 protein-coding genes and 66 noncoding RNA loci are differentially expressed (“patterned”) across the layers, on the basis of a machine-learning model (naive Bayes) approach. Layers 2-6b are each associated with specific functional and disease annotations that provide insights into their biological roles. This new resource (http://genserv.anat.ox.ac.uk/layers) greatly extends currently available resources, such as the Allen Mouse Brain Atlas and microarray data sets, by providing quantitative expression levels, by being genome-wide, by including novel loci, and by identifying candidate alternatively spliced transcripts that are differentially expressed across layers.

Long noncoding RNA genes: conservation of sequence and brain expression among diverse amniotes

BackgroundLong considered to be the building block of life, it is now apparent that protein is only one of many functional products generated by the eukaryotic genome. Indeed, more of the human genome is transcribed into noncoding sequence than into protein-coding sequence. Nevertheless, whilst we have developed a deep understanding of the relationships between evolutionary constraint and function for protein-coding sequence, little is known about these relationships for non-coding transcribed sequence. This dearth of information is partially attributable to a lack of established non-protein-coding RNA (ncRNA) orthologs among birds and mammals within sequence and expression databases.ResultsHere, we performed a multi-disciplinary study of four highly conserved and brain-expressed transcripts selected from a list of mouse long intergenic noncoding RNA (lncRNA) loci that generally show pronounced evolutionary constraint within their putative promoter regions and across exon-intron boundaries. We identify some of the first lncRNA orthologs present in birds (chicken), marsupial (opossum), and eutherian mammals (mouse), and investigate whether they exhibit conservation of brain expression. In contrast to conventional protein-coding genes, the sequences, transcriptional start sites, exon structures, and lengths for these non-coding genes are all highly variable.ConclusionsThe biological relevance of lncRNAs would be highly questionable if they were limited to closely related phyla. Instead, their preservation across diverse amniotes, their apparent conservation in exon structure, and similarities in their pattern of brain expression during embryonic and early postnatal stages together indicate that these are functional RNA molecules, of which some have roles in vertebrate brain development.

Mutation of the Variant α-Tubulin TUBA8 Results in Polymicrogyria with Optic Nerve Hypoplasia

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

The Subventricular Zone Is the Developmental Milestone of a 6-Layered Neocortex: Comparisons in Metatherian and Eutherian Mammals

The major lineages of mammals (Eutheria, Metatheria, and Monotremata) diverged more than 100 million years ago and have undergone independent changes in the neocortex. We found that adult South American gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii) possess a significantly lower number of cerebral cortical neurons compared with the mouse (Mus musculus). To determine whether the difference is reflected in the development of the cortical germinal zones, the location of progenitor cell divisions was examined in opossum, tammar wallaby, and rat. The basic pattern of the cell divisions was conserved, but the emergence of a distinctive band of dividing cells in the subventricular zone (SVZ) occurred relatively later in the opossum (postnatal day [P14]) and the tammar wallaby (P40) than in rodents. The planes of cell divisions in the ventricular zone (VZ) were similar in all species, with comparable mRNA expression patterns of Brn2, Cux2, NeuroD6, Tbr2, and Pax6 in opossum (P12 and P20) and mouse (embryonic day 15 and P0). In conclusion, the marsupial neurodevelopmental program utilizes an organized SVZ, as indicated by the presence of intermediate (or basal) progenitor cell divisions and gene expression patterns, suggesting that the SVZ emerged prior to the Eutherian-Metatherian split.

Cross-talking noncoding RNAs contribute to cell-specific neurodegeneration in SCA7.

What causes the tissue-specific pathology of diseases resulting from mutations in housekeeping genes? Specifically, in spinocerebellar ataxia type 7 (SCA7), a neurodegenerative disorder caused by a CAG-repeat expansion in ATXN7 (which encodes an essential component of the mammalian transcription coactivation complex, STAGA), the factors underlying the characteristic progressive cerebellar and retinal degeneration in patients were unknown. We found that STAGA is required for the transcription initiation of miR-124, which in turn mediates the post-transcriptional cross-talk between lnc-SCA7, a conserved long noncoding RNA, and ATXN7 mRNA. In SCA7, mutations in ATXN7 disrupt these regulatory interactions and result in a neuron-specific increase in ATXN7 expression. Strikingly, in mice this increase is most prominent in the SCA7 disease-relevant tissues, namely the retina and cerebellum. Our results illustrate how noncoding RNA–mediated feedback regulation of a ubiquitously expressed housekeeping gene may contribute to specific neurodegeneration.

Extensive microRNA-mediated crosstalk between lncRNAs and mRNAs in mouse embryonic stem cells

Recently, a handful of intergenic long noncoding RNAs (lncRNAs) have been shown to compete with mRNAs for binding to miRNAs and to contribute to development and disease. Beyond these reports, little is yet known of the extent and functional consequences of miRNA-mediated regulation of mRNA levels by lncRNAs. To gain further insight into lncRNA-mRNA miRNA-mediated crosstalk, we reanalyzed transcriptome-wide changes induced by the targeted knockdown of over 100 lncRNA transcripts in mouse embryonic stem cells (mESCs). We predicted that, on average, almost one-fifth of the transcript level changes induced by lncRNAs are dependent on miRNAs that are highly abundant in mESCs. We validated these findings experimentally by temporally profiling transcriptome-wide changes in gene expression following the loss of miRNA biogenesis in mESCs. Following the depletion of miRNAs, we found that >50% of lncRNAs and their miRNA-dependent mRNA targets were up-regulated coordinately, consistent with their interaction being miRNA-mediated. These lncRNAs are preferentially located in the cytoplasm, and the response elements for miRNAs they share with their targets have been preserved in mammals by purifying selection. Lastly, miRNA-dependent mRNA targets of each lncRNA tended to share common biological functions. Post-transcriptional miRNA-mediated crosstalk between lncRNAs and mRNA, in mESCs, is thus surprisingly prevalent, conserved in mammals, and likely to contribute to critical developmental processes.

Complexities of post-transcriptional regulation and the modeling of ceRNA crosstalk

ABSTRACT Control of gene and protein expression is required for cellular homeostasis and is disrupted in disease. Following transcription, mRNA turnover and translation is modulated, most notably by microRNAs (miRNAs). This modulation is controlled by transcriptional and post-transcriptional events that alter the availability of miRNAs for target binding. Recent studies have proposed that some transcripts – termed competitive endogenous RNAs (ceRNAs) – sequester a miRNA and diminish its repressive effects on other transcripts. Such ceRNAs thus mutually alter each other’s abundance by competing for binding to a common set of miRNAs. Some question the relevance of ceRNA crosstalk, arguing that an individual transcript, when its abundance lies within a physiological range of gene expression, will fail to compete for miRNA binding due to the high abundance of other miRNA binding sites across the transcriptome. Despite this, some experimental evidence is consistent with the ceRNA hypothesis. In this review, we draw upon existing data to highlight mechanistic and theoretical aspects of ceRNA crosstalk. Our intent is to propose how understanding of ceRNA crosstalk mechanisms can be improved and what evidence is required to demonstrate a ceRNA mechanism. A greater understanding of factors affecting ceRNA crosstalk should shed light on its relevance in physiological states. Graphical Abstract

Insights into the post-transcriptional regulation of the mitochondrial electron transport chain

The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction.

Cerox1 and microRNA-488-3p noncoding RNAs jointly regulate mitochondrial complex I catalytic activity

To generate energy efficiently, the cell is uniquely challenged to co-ordinate the abundance of electron transport chain protein subunits expressed from both nuclear and mitochondrial genomes. How an effective stoichiometry of this many constituent subunits is co-ordinated post-transcriptionally remains poorly understood. Here we show that Cerox1, an unusually abundant cytoplasmic long noncoding RNA (lncRNA), modulates the levels of mitochondrial complex I subunit transcripts in a manner that requires binding to microRNA-488-3p. Increased abundance of Cerox1 cooperatively elevates complex I subunit protein abundance and enzymatic activity, decreases reactive oxygen species production, and protects against the complex I inhibitor rotenone. Cerox1 function is conserved across placental mammals: human and mouse orthologues effectively modulate complex I enzymatic activity in mouse and human cells, respectively. Cerox1 is the first lncRNA demonstrated, to our knowledge, to regulate mitochondrial oxidative phosphorylation (OXPHOS) and, with miR-488-3p, represent novel targets for the modulation of complex I activity.

The long non‐coding RNA Paupar promotes KAP1‐dependent chromatin changes and regulates olfactory bulb neurogenesis

Many long non‐coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and mechanisms of action remain poorly understood. Paupar, a CNS‐expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of transcriptional regulatory elements in a genome‐wide manner. We show here that the Paupar lncRNA directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through the formation of a ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar‐KAP1 genome‐wide co‐occupancy reveals a fourfold enrichment of overlap between Paupar and KAP1 bound sequences, the majority of which also appear to associate with PAX6. Furthermore, both Paupar and Kap1 loss‐of‐function in vivo disrupt olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans‐acting modes of lncRNA‐mediated epigenetic regulation and the mechanisms of KAP1 genomic recruitment, and identify Paupar and Kap1 as regulators of neurogenesis in vivo.