A novel antisense lncRNA, ARBAG harboring an RNA destabilizing GWAS variant for C-peptide dictates the transcript isoforms of GABRA6 in cerebellum.

Human disease-associated genetic variations often map to long non-coding RNA (lncRNA) genes; however, elucidation of their functional impact is challenging. We previously identified a new genetic variant rs4454083 (A/G) residing in exon of an uncharacterized lncRNA ARBAG that strongly associates with plasma levels of C-peptide, a hormone that regulates insulin bioavailability. On the opposite strand, rs4454083 also corresponds to an intron of a cerebellum-specific GABA receptor subunit gene GABRA6 that mediates strengthening of inhibitory synapses by insulin. Here, we show that alleles of rs4454083 modulate transcript levels of the antisense gene, ARBAG, which then controls the expression of the sense gene, GABRA6. Predisposing to low C-peptide, GG (a minor allele genotype across ethnicities) stabilizes ARBAG lncRNA causing higher transcript levels in cerebellum. ARBAG lncRNA abundance leads to cleavage of GABRA6 mRNA at the complementary region, resulting in a dysfunctional GABRA6 protein that would not be recruited for synapse strengthening. Together, our findings in human cerebellar cell-line and induced Pluripotent Stem Cells (iPSCs) demonstrate biological role of a novel lncRNA in determining the ratio of mRNA isoforms of a protein-coding gene and the ability of an embedded variant in modulating lncRNA stability leading to inter-individual differences in protein expression.

Neev, a novel long non-coding RNA, is expressed in chaetoblasts during regeneration of Eisenia fetida.

Eisenia fetida, the common vermicomposting earthworm, shows robust regeneration of posterior segments removed by amputation. During the period of regeneration, the newly formed tissue initially contains only undifferentiated cells but subsequently differentiates into a variety of cell types including muscle, nerve and vasculature. Transcriptomics analysis, reported previously, provided a number of candidate non-coding RNAs that were induced during regeneration. We found that one such long non-coding RNA (lncRNA) is expressed in the skin, only at the base of newly formed chaetae. The spatial organization and precise arrangement of the regenerating chaetae and the cells expressing the lncRNA on the ventral side clearly support a model wherein the regenerating tissue contains a zone of growth and cell division at the tip and a zone of differentiation at the site of amputation. The temporal expression pattern of the lncRNA, named Neev, closely resembled the pattern of chitin synthase genes, implicated in chaetae formation. We found that the lncRNA has 49 sites for binding a set of four microRNAs (miRNAs) while the chitin synthase 8 mRNA has 478 sites. The over-representation of shared miRNA sites suggests that lncRNA Neev may act as a miRNA sponge to transiently de-repress chitin synthase 8 during formation of new chaetae in the regenerating segments of Eisenia fetida.

Parentally inherited long non-coding RNA Cyrano is involved in zebrafish neurodevelopment.

Transfer of genetic material from parents to progeny via fusion of gametes is a way to ensure flow of information from one generation to the next. Apart from the genetic material, gametes provide a rich source of other factors such as RNA and proteins which can control traits of the embryo. Non-coding RNAs are not only carriers of regulatory information but can also encode memory of events of parental life. Here, we explore the possibility of parental inheritance of non-coding RNAs, especially long non-coding RNAs. Meta-analysis of RNA-seq data revealed several non-coding RNAs present in zebrafish oocyte, sperm and 2cell-stage. The embryo is transcriptionally silent at this stage, we rationalize that all the RNAs detectable at 2cell-stage are deposited either by sperm or oocyte or both and thus inherited. In the inherited pool, we noticed a conserved lncRNA, Cyrano previously known for zebrafish brain development. Knockdown of inherited Cyrano by miR-7 without changing zygotic Cyrano altered brain morphology at 24 hpf and 48 hpf. This defect could be partially rescued by injecting full length Cyrano lncRNA or a mutant resilient to knock-down by miR-7. In future, there is ample scope to check the possibility of inherited lncRNAs as carriers of memory of parental life events and building blocks that set up an initial platform for development.

Identification of novel circadian transcripts in the zebrafish retina.

High fecundity, transparent embryos for monitoring the rapid development of organs and the availability of a well-annotated genome has made zebrafish a model organism of choice for developmental biology and neurobiology. This vertebrate model, which is also a favourite in chronobiology studies, shows striking circadian rhythmicity in behaviour. Here, we identify novel genes in the zebrafish genome that are expressed in the zebrafish retina. We further resolve the expression pattern over time and tentatively assign specific novel transcripts to retinal bipolar cells of the inner nuclear layer. Using chemical ablation and free run experiments, we segregate the transcripts that are rhythmic when entrained by light from those that show sustained oscillations in the absence of external cues. The transcripts reported here with rigorous annotation and specific functions in circadian biology provide the groundwork for functional characterization of novel players in the zebrafish retinal clock.

microRNA dysregulation in polyglutamine toxicity of TATA-box binding protein is mediated through STAT1 in mouse neuronal cells.

BACKGROUND: Polyglutamine diseases constitute a class of neurodegenerative disorders associated with expansion of the cytosine-adenine-guanine (CAG) triplet, in protein coding genes. Expansion of a polyglutamine tract in the N-terminal of TBP is the causal mutation in SCA17. Brain sections of patients with spinocerebellar ataxia 17 (SCA17), a type of neurodegenerative disease, have been reported to contain protein aggregates of TATA-binding protein (TBP). It is also implicated in other neurodegenerative diseases like Huntington's disease, since the protein aggregates formed in such diseases also contain TBP. Dysregulation of miR-29a/b is another common feature of neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and SCA17. Using a cellular model of SCA17, we identified key connections in the molecular pathway from protein aggregation to miRNA dysregulation. METHODS: Gene expression profiling was performed subsequent to the expression of TBP containing polyglutamine in a cellular model of SCA17. We studied the expression of STAT1 and other interferon-gamma dependent genes in neuronal apoptosis. The molecular pathway leading to the dysregulation of miRNA in response of protein aggregation and interferon release was investigated using RNAi-mediated knockdown of STAT1. RESULTS: We show that the accumulation of polyglutamine-TBP in the cells results in interferon-gamma release which in turn signals through STAT1 leading to downregulation of miR-29a/b. We propose that the release of interferons by cells harboring toxic protein aggregates may trigger a bystander effect resulting in loss of neurons. Interferon-gamma also led to upregulation of miR-322 although this effect is not mediated through STAT1. CONCLUSIONS: Our investigation shows that neuroinflammation could be an important player in mediating the transcriptional dysregulation of miRNA and the subsequent apoptotic effect of toxic polyglutamine-TBP. The involvement of immunomodulators in polyglutamine diseases holds special therapeutic relevance in the light of recent findings that interferon-gamma can modulate behavior.

Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice.

Several microRNAs have been implicated in neurogenesis, neuronal differentiation, neurodevelopment, and memory. Development of miRNA-based therapeutics, however, needs tools for effective miRNA modulation, tissue-specific delivery, and in vivo evidence of functional effects following the knockdown of miRNA. Expression of miR-29a is reduced in patients and animal models of several neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, and spinocerebellar ataxias. The temporal expression pattern of miR-29b during development also correlates with its protective role in neuronal survival. Here, we report the cellular and behavioral effect of in vivo, brain-specific knockdown of miR-29. We delivered specific anti-miRNAs to the mouse brain using a neurotropic peptide, thus overcoming the blood-brain-barrier and restricting the effect of knockdown to the neuronal cells. Large regions of the hippocampus and cerebellum showed massive cell death, reiterating the role of miR-29 in neuronal survival. The mice showed characteristic features of ataxia, including reduced step length. However, the apoptotic targets of miR-29, such as Puma, Bim, Bak, or Bace1, failed to show expected levels of up-regulation in mice, following knockdown of miR-29. In contrast, another miR-29 target, voltage-dependent anion channel1 (VDAC1), was found to be induced several fold in the hippocampus, cerebellum, and cortex of mice following miRNA knockdown. Partial restoration of apoptosis was achieved by down-regulation of VDAC1 in miR-29 knockdown cells. Our study suggests that regulation of VDAC1 expression by miR-29 is an important determinant of neuronal cell survival in the brain. Loss of miR-29 results in dysregulation of VDAC1, neuronal cell death, and an ataxic phenotype.

Novel MicroRNA signatures in HPV-mediated cervical carcinogenesis in Indian women.

This study aimed to investigate the role of miRNAs in HPV-mediated cervical pre-cancer and cancer cases in Indian population. We analysed the HPV infection and its genotypes in uterine cervical pre-cancer (n = 80), cancer (n = 200) and normal cervical samples (n = 150) by consensus sequence PCR followed by type specific PCRs. Also, microRNA profiling was done in a subset of cervical pre-cancer (n = 20), cancer cases (n = 50) and normal samples (n = 30) by real-time quantitative PCR (qRT-PCR). The prevalence of HPV infection in pre-cancer was found to be 81 % (65/80) and 94 % (188/200) in cancer cases, with most predominant high-risk HPV type-16 (HR-HPV-16) in 83 % of cancer and 91 % of pre- cancer cases, respectively. Whereas in controls, the HPV infection was found to be very low (5 %). The miRNA profiling revealed that in cervical pre-cancer, 100 miRNAs were significantly (p < 0.001) differentially expressed with 70 miRNAs upregulated and 30 miRNAs downregulated. In cervical cancer cases, 383 miRNA were found to be differentially expressed (p < 0.001), of which 350 miRNAs were upregulated and 33 miRNAs were downregulated. We also observed that 182 miRNAs were differentially expressed (p < 0.001) in HPV-16/18-positive (SiHa/HeLa) cell lines compared with HPV-negative (C33A) cell line. In addition, we identified the novel microRNAs such as miR-892b, miR-500, miR-888, miR-505 and miR-711 in cervical precancerous lesions and cervical cancer cases in Indian population. Taken together, the study demonstrates a crucial role of microRNAs in cervical cancer, which may serve as potential early diagnostic markers for cervical carcinogenesis.

miR-34 is maternally inherited in Drosophila melanogaster and Danio rerio.

MicroRNAs (miRNAs) are small, endogenous, regulatory RNA molecules that can bind to partially complementary regions on target messenger RNAs and impede their expression or translation. We rationalized that miRNAs, being localized to the cytoplasm, will be maternally inherited during fertilization and may play a role in early development. Although Dicer is known to be essential for the transition from single-celled zygote to two-cell embryo, a direct role for miRNAs has not yet been demonstrated. We identified miRNAs with targets in zygotically expressed transcripts in Drosophila using a combination of transcriptome analysis and miRNA target prediction. We experimentally established that Drosophila miRNA dme-miR-34, the fly homologue of the cancer-related mammalian miRNA miR-34, involved in somatic-cell reprogramming and having critical role in early neuronal differentiation, is present in Drosophila embryos before initiation of zygotic transcription. We also show that the Drosophila miR-34 is dependent on maternal Dicer-1 for its expression in oocytes. Further, we show that miR-34 is also abundant in unfertilized oocytes of zebrafish. Its temporal expression profile during early development showed abundant expression in unfertilized oocytes that gradually decreased by 5 days post-fertilization (dpf). We find that knocking down the maternal, but not the zygotic, miR-34 led to developmental defects in the neuronal system during early embryonic development in zebrafish. Here, we report for the first time, the maternal inheritance of an miRNA involved in development of the neuronal system in a vertebrate model system.

Comprehensive expression analyses of neural cell-type-specific miRNAs identify new determinants of the specification and maintenance of neuronal phenotypes.

MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions. However, a systematic analysis of brain miRNA functions has been hindered by a lack of comprehensive information regarding the distribution of miRNAs in neuronal versus glial cells. To address this issue, we performed microarray analyses of miRNA expression in the four principal cell types of the CNS (neurons, astrocytes, oligodendrocytes, and microglia) using primary cultures from postnatal d 1 rat cortex. These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types. We also demonstrate that individual neuron-enriched or neuron-diminished RNAs had a significant impact on the specification of neuronal phenotype: overexpression of the neuron-enriched miRNAs miR-376a and miR-434 increased the differentiation of neural stem cells into neurons, whereas the opposite effect was observed for the glia-enriched miRNAs miR-223, miR-146a, miR-19, and miR-32. In addition, glia-enriched miRNAs were shown to inhibit aberrant glial expression of neuronal proteins and phenotypes, as exemplified by miR-146a, which inhibited neuroligin 1-dependent synaptogenesis. This study identifies new nervous system functions of specific miRNAs, reveals the global extent to which the brain may use differential miRNA expression to regulate neural cell-type-specific phenotypes, and provides an important data resource that defines the compartmentalization of brain miRNAs across different cell types.

A study on the influence of different promoter and 5'UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene ß glucuronidase in tobacco and cotton.

Several reports of promoters from plants, viral and artificial origin that confer high constitutive expression are known. Among these the CaMV 35S promoter is used extensively for transgene expression in plants. We identified candidate promoters from Arabidopsis based on their transcript levels (meta-analysis of available microarray control datasets) to test their activity in comparison to the CaMV 35S promoter. A set of 11 candidate genes were identified which showed high transcript levels in the aerial tissue (i.e. leaf, shoot, flower and stem). In the initial part of the study binary vectors were developed wherein the promoter and 5'UTR region of these candidate genes (Upstream Regulatory Module, URM) were cloned upstream to the reporter gene ß glucuronidase (gus). The promoter strengths were tested in transformed callus of Nicotiana tabacum and Gossypium hirsutum. On the basis of the results obtained from the callus, the influence of the URM cassettes on transgene expression was tested in transgenic tobacco. The URM regions of the genes encoding a subunit of photosystem I (PHOTO) and geranyl geranyl reductase (GGR) in A. thaliana genome showed significantly high levels of GUS activity in comparison to the CaMV 35S promoter. Further, when the 5'UTRs of both the genes were placed downstream to the CaMV 35S promoter it led to a substantial increase in GUS activity in transgenic tobacco lines and cotton callus. The enhancement observed was even higher to that observed with the viral leader sequences like Ω and AMV, known translational enhancers. Our results indicate that the two URM cassettes or the 5'UTR regions of PHOTO and GGR when placed downstream to the CaMV 35S promoter can be used to drive high levels of transgene expression in dicotyledons.

Proximity of H2A.Z containing nucleosome to the transcription start site influences gene expression levels in the mammalian liver and brain.

Nucleosome positioning maps of several organisms have shown that Transcription Start Sites (TSSs) are marked by nucleosome depleted regions flanked by strongly positioned nucleosomes. Using genome-wide nucleosome maps and histone variant occupancy in the mouse liver, we show that the majority of genes were associated with a single prominent H2A.Z containing nucleosome in their promoter region. We classified genes into clusters depending on the proximity of H2A.Z to the TSS. The genes with no detectable H2A.Z showed lowest expression level, whereas H2A.Z was positioned closer to the TSS of genes with higher expression levels. We confirmed this relation between the proximity of H2A.Z and expression level in the brain. The proximity of histone variant H2A.Z, but not H3.3 to the TSS, over seven consecutive nucleosomes, was correlated with expression. Further, a nucleosome was positioned over the TSS of silenced genes while it was displaced to expose the TSS in highly expressed genes. Our results suggest that gene expression levels in vivo are determined by accessibility of the TSS and proximity of H2A.Z.

Noncoding RNAs: Emerging regulators of behavioral complexity.

The mammalian genome encodes thousands of non-coding RNAs (ncRNAs), ranging in size from about 20 nucleotides (microRNAs or miRNAs) to kilobases (long non-coding RNAs or lncRNAs). ncRNAs contribute to a layer of gene regulation that could explain the evolution of massive phenotypic complexity even as the number of protein-coding genes remains unaltered. We propose that low conservation, poor expression, and highly restricted spatiotemporal expression patterns-conventionally considered ncRNAs may affect behavior through direct, rapid, and often sustained regulation of gene expression at the transcriptional, post-transcriptional, or translational levels. Besides these direct roles, their effect during neurodevelopment may manifest as behavioral changes later in the organism's life, especially when exposed to environmental cues like stress and seasonal changes. The lncRNAs affect behavior through diverse mechanisms like sponging of miRNAs, recruitment of chromatin modifiers, and regulation of alternative splicing. We highlight the need for synthesis between rigorously designed behavioral paradigms in model organisms and the wide diversity of behaviors documented by ethologists through field studies on organisms exquisitely adapted to their environmental niche. Comparative genomics and the latest advancements in transcriptomics provide an unprecedented scope for merging field and lab studies on model and non-model organisms to shed light on the role of ncRNAs in driving the behavioral responses of individuals and groups. We touch upon the technical challenges and contentious issues that must be resolved to fully understand the role of ncRNAs in regulating complex behavioral traits. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

A Novel Cis-Regulatory lncRNA, Kalnc2, Downregulates Kalrn Protein-Coding Transcripts in Mouse Neuronal Cells.

The KALRN gene encodes several multi-domain protein isoforms that localize to neuronal synapses, conferring the ability to grow and retract dendritic spines and shaping axonal outgrowth, dendrite morphology, and dendritic spine re-modeling. The KALRN genomic locus is implicated in several neurodevelopmental and neuropsychiatric diseases, including autism, schizophrenia, bipolar disease, and intellectual disability. We have previously shown that a novel brain-specific long non-coding RNA (lncRNA) arising from the 5' end of the kalrna gene, called durga, regulates neuronal morphology in zebrafish. Here, we characterized mammalian Kalrn loci, annotating and experimentally validating multiple novel non-coding RNAs, including linear and circular variants. Comparing the mouse and human loci, we show that certain non-coding RNAs and Kalrn protein-coding isoforms arising from the locus show similar expression dynamics during development. In humans, mice, and zebrafish, the 5' end of the Kalrn locus gives rise to a chromatin-associated lncRNA that is present in adult ovaries, besides being expressed during brain development and enriched in certain regions of the adult brain. Ectopic expression of this lncRNA led to the downregulation of all the major Kalrn mRNA isoforms. We propose that this lncRNA arising from the 5' end of the Kalrn locus is functionally the mammalian ortholog of zebrafish lncRNA durga.

Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis.

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.

Consensus miRNA expression profiles derived from interplatform normalization of microarray data.

Eukaryotic gene expression is controlled at the post-transcriptional level by small noncoding RNAs called microRNAs (miRNA). miRNAs play important roles during early development and participate in gene regulatory circuits in the cell. Different high-throughput expression analysis methods including microarrays, bead-based detection, and small RNA cloning have been applied to quantitatively detect miRNAs in various tissues, cell types, and biological conditions. High-throughput expression data was collected from public repositories and processed to create a database of miRNA expression profiles. Several commonly used normalization methods were compared to identify suitable methods for cross-platform comparison of high-throughput miRNA expression data. The database provides interlaboratory and interplatform validated reference expression levels for miRNAs. The normalized expression profiles were validated by querying for well-established features of miRNA expression. Firstly, expression profiles of several tissue-specific miRNAs showed good agreement between the database and previously reported profiles. We have also identified a set of miRNAs that are constitutively expressed across mammalian tissues. Secondly, we used the database to compare the expression patterns of miRNAs belonging to the let-7 family, where the divergence in expression patterns implies that they may have diversified functionally. Lastly, we compared expression profiles of intronic and clustered miRNAs. Expression profiles of intronic miRNAs and clustered miRNAs showed either very good, or in certain cases, very poor correlation with the host gene. Interplatform comparison of miRNA expression profiles thus provides a resource of consensus expression profiles that can be used in the future for studying miRNA function and regulation.

Regulation of BACE1 by miR-29a/b in a cellular model of Spinocerebellar Ataxia 17.

Polyglutamine diseases are a class of neurodegenerative disorders characterized by expansion of polyglutamine repeats, protein aggregation and neuronal cell death in specific regions of the brain. The expansion of a polyglutamine repeat in the TATA binding protein (TBP) causes a neurodegenerative disease, Spinocerebellar Ataxia 17 (SCA17). This disease is characterized by intranuclear protein aggregates and selective loss of cerebellar neurons, including Purkinje cells. MicroRNAs are small, endogenous, regulatory non-coding RNA molecules that bind to messenger RNAs with partial complementarity and interfere in their expression. Here, we used a cellular model of SCA17 where we expressed TBP with 16 (normal) or 59 (pathogenic) polyglutamines and found differential expression of several microRNAs. Specifically, we found two microRNAs, miR-29a/b, were down-regulated. With miR-29a/b down regulation, we found an increased expression of targets of miR-29a/b -beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), p53 upregulated modulator of apoptosis (PUMA) and BAK, increased cytochrome c release and apoptosis. Restoration of miR-29a/b in the pathogenic polyglutamine background reduced the BACE1expression. While, antagomiRs against miR-29a/b resulted in an increase in BACE1 levels and neuronal apoptosis. In spite of the elevation of BACE1 in Alzhemiers disease, its role in neuronal cell death has not been established. Here, we show that increased BACE1 expression is not sufficient to cause apoptosis. However restoring level of BACE1 to normal in polyglutamine cells partially reduced neuronal apoptosis. We show a role for the miR-29a/b-BACE1 regulatory interaction in SCA17, suggesting that this microRNA could be part of a common molecular mechanism leading to neuronal cell death in multiple neurodegenerative disorders. The identification of a common mechanism of microRNA mediated neurodegeneration not only improves our understanding of the process, but also provides promising and novel therapeutic targets.

Early life stressful experiences escalate aggressive behavior in adulthood via changes in transthyretin expression and function.

Escalated and inappropriate levels of aggressive behavior referred to as pathological in psychiatry can lead to violent outcomes with detrimental impact on health and society. Early life stressful experiences might increase the risk of developing pathological aggressive behavior in adulthood, though molecular mechanisms remain elusive. Here, we provide prefrontal cortex and hypothalamus specific transcriptome profiles of peripubertal stress (PPS) exposed Balb/c adult male mice exhibiting escalated aggression and adult female mice resilient to such aberrant behavioral responses. We identify transthyretin (TTR), a well known thyroid hormone transporter, as a key regulator of PPS induced escalated aggressive behavior in males. Brain-region-specific long-term changes in Ttr gene expression and thyroid hormone (TH) availability were evident in PPS induced escalated aggressive male mice, circulating TH being unaltered. Ttr promoter methylation marks were also altered being hypermethylated in hypothalamus and hypomethylated in prefrontal cortex corroborating with its expression pattern. Further, Ttr knockdown in hypothalamus resulted in escalated aggressive behavior in males without PPS and also reduced TH levels and expression of TH-responsive genes (Nrgn, Trh, and Hr). Escalated aggressive behavior along with reduced Ttr gene expression and TH levels in hypothalamus was also evident in next generation F1 male progenies. Our findings reveal that stressful experiences during puberty might trigger lasting escalated aggression by modulating TTR expression in brain. TTR can serve as a potential target in reversal of escalated aggression and related psychopathologies.

Incomplete penetrance and variable expressivity: is there a microRNA connection?

Incomplete penetrance and variable expressivity are non-Mendelian phenomena resulting in the lack of correlation between genotype and phenotype. Not withstanding the diversity in mechanisms, differential expression of homologous alleles within cells manifests as variations in penetrance and expressivity of mutations between individuals of the same genotype. These phenomena are seen most often in dominantly inherited diseases, implying that they are sensitive to concentration of the gene product. In this framework and the advances in understanding the role of microRNA (miRNA) in fine-tuning gene expression at translational level, we propose miRNA-mediated regulation as a mechanism for incomplete penetrance and variable expressivity. The presence of miRNA binding sites at 3' UTR, co-expression of target gene-miRNA pairs for genes showing incomplete penetrance and variable expressivity derived from available data lend support to our hypothesis. Single nucleotide polymorphisms in the miRNA target site facilitate the implied differential targeting of the transcripts from homologous alleles.

Multiple Alu Exonization in 3'UTR of a Primate-Specific Isoform of CYP20A1 Creates a Potential miRNA Sponge.

Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3'UTR. CYP20A1_Alu-LT, confirmed by 3'RACE, is an outlier in length (9 kb 3'UTR) and widely expressed. Using publically available data sets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15,928 human cortical neurons. miRanda predicts ∼4,700 miRNA recognition elements (MREs) for ∼1,000 miRNAs, primarily originated within these 3'UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. In total, 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared with random sets of differentially expressed genes (P = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA-mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.