Analyses of circRNA Expression throughout the Light-Dark Cycle Reveal a Strong Regulation of Cdr1as, Associated with Light Entrainment in the SCN

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that Cdr1as, a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, Cdr1as has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that Cdr1as is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of Cdr1as in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation

Analyses of circRNA expression throughout the light-dark cycle reveal a strong regulation of (Cdr1as), associated with light entrainment in the SCN

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that (Cdr1as), a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, (Cdr1as) has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that (Cdr1as) is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of (Cdr1as) in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation

Inter-chromosomal insertions at Xq27.1 associated with retinal dystrophy induce dysregulation of LINC00632 and CDR1as/ciRS-7.

In two unrelated families with X-linked inherited retinal dystrophy, identification of the causative variants was elusive. Interrogation of the next-generation sequencing (NGS) data revealed a dark intergenic region on Xq27.1 with poor coverage. Long-range PCR and DNA walking across this region revealed different inter-chromosomal insertions into the human-specific palindrome on Xq27.1: a 58 kb insertion of 9p24.3 [der(X)dir ins(X;9)(q27.1;p24.3)] in family 1 and a 169 kb insertion of 3p14.2 [der(X)inv ins(X;3)(q27.1;p14.2)] in family 2. To explore the functional consequence of these structural variants in genomic and cellular contexts, induced pluripotent stem cells were derived from affected and control fibroblasts and differentiated to retinal organoids (ROs) and retinal pigment epithelium. Transcriptional dysregulation was evaluated using RNA sequencing (RNA-seq) and RT-qPCR. A downstream long non-coding RNA, LINC00632 (Xq27.1), was upregulated in ROs from both families compared to control samples. In contrast, the circular RNA CDR1as/ciRS-7 (circular RNA sponge for miR-7), spliced from linear LINC00632, was downregulated. To investigate this tissue-specific dysregulation, we interrogated the landscape of the locus using Hi-C and cleavage under targets and tagmentation sequencing (CUT&Tag). This revealed active retinal enhancers within the insertions within a topologically associated domain that also contained the upstream promoter of LINC00632, permitting ectopic contact. Furthermore, CDR1as/ciRS-7 acts as a sponge for miR-7, and target genes of miR-7 were also dysregulated in ROs derived from both families. We describe a new genomic mechanism for retinal dystrophy, and our data support a convergent tissue-specific mechanism of altered regulation of LINC00632 and CDR1as/ciRS-7 as a consequence of the insertions within the palindrome on Xq27.1

Mathematical model of a fountain with a water picture in the shape of an hourglass

The work presents a mathematical model of a fountain providing an hourglass-shaped water image based on four of its basic parameters: height H, coefficient of contraction χ, diameter of n nozzles arrangement Dna, and coefficient of contraction location σ. The developed model makes possible to determine the angle α specifying the slope of the nozzles to the horizontal plane and determining the remaining parameters of the fountain, especially its outer diameter Df as well as the diameter of the position of the stream tops Dmax. The developed model has been implemented in an Excel spreadsheet, which enables quick conversion of desired water images. The developed model works for n≤24 nozzles, however, derived dependencies allow for a quick extension of the model for n>24

Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease

In the past decade, single-cell technologies have proliferated and improved from their technically challenging beginnings to become common laboratory methods capable of determining the expression of thousands of genes in thousands of cells simultaneously. The field has progressed by taking the CNS as a primary research subject - the cellular complexity and multiplicity of neuronal cell types provide fertile ground for the increasing power of single-cell methods. Current single-cell RNA sequencing methods can quantify gene expression with sufficient accuracy to finely resolve even subtle differences between cell types and states, thus providing a great tool for studying the molecular and cellular repertoire of the CNS and its disorders. However, single-cell RNA sequencing requires the dissociation of tissue samples, which means that the interrelationships between cells are lost. Spatial transcriptomic methods bypass tissue dissociation and retain this spatial information, thereby allowing gene expression to be assessed across thousands of cells within the context of tissue structural organization. Here, we discuss how single-cell and spatially resolved transcriptomics have been contributing to unravelling the pathomechanisms underlying brain disorders. We focus on three areas where we feel these new technologies have provided particularly useful insights: selective neuronal vulnerability, neuroimmune dysfunction and cell-type-specific treatment response. We also discuss the limitations and future directions of single-cell and spatial RNA sequencing technologies

Mathematical model of a fountain with a water picture in the shape of an hourglass

The work presents a mathematical model of a fountain providing an hourglass-shaped water image based on four of its basic parameters: height H, coefficient of contraction χ, diameter of n nozzles arrangement Dna, and coefficient of contraction location σ. The developed model makes possible to determine the angle α specifying the slope of the nozzles to the horizontal plane and determining the remaining parameters of the fountain, especially its outer diameter Df as well as the diameter of the position of the stream tops Dmax. The developed model has been implemented in an Excel spreadsheet, which enables quick conversion of desired water images. The developed model works for n≤24 nozzles, however, derived dependencies allow for a quick extension of the model for n&gt;24.</jats:p

RNA regulation in brain function and disease 2022 (NeuroRNA): A conference report

Recent research integrates novel technologies and methods from the interface of RNA biology and neuroscience. This advancing integration of both fields creates new opportunities in neuroscience to deepen the understanding of gene expression programs and their regulation that underlies the cellular heterogeneity and physiology of the central nervous system. Currently, transcriptional heterogeneity can be studied in individual neural cell types in health and disease. Furthermore, there is an increasing interest in RNA technologies and their application in neurology. These aspects were discussed at an online conference that was shortly named NeuroRNA.</jats:p

Single-molecule fluorescence in situ hybridization (FISH) of circular RNA CDR1as

Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus