Activity-dependent regulation of miRNAs in different subcellular compartments of neurons and its implications for neuronal morphogenesis and plasticity

The activity-dependent spatiotemporal regulation of gene expression in neurons is essential for the formation and function of neuronal circuits within the brain. Recently microRNAs, a new class of post-transcriptional regulators of gene expression were implicated in the regulation of neuronal differentiation and development. Furthermore, in mature fully developed neurons, miRNAs (e.g. miR-134) were shown to be involved in the control of local protein synthesis in the vicinity of dendritic spines (Schratt et al., 2006). Activity-dependent local protein synthesis is required for synaptic plasticity, which is believed to be one of the molecular substrates of learning and memory. Nonetheless, the molecular mechanisms underlying the function and regulation of miRNAs during synaptic plasticity are poorly understood. In a previous publication from our lab, it was shown that the activity of the brain-enriched miRNA - miR-134 is regulated by brain-derived neurotrophic factor, which is released upon synaptic stimulation in neurons (Schratt et al, 2006). Interestingly, in the mouse genome this miRNA is encoded in a large miRNA cluster (miR379-410 cluster) consisting of 39 miRNAs. The expression of the miR379-410 cluster is induced upon neuronal activity by virtue of myocyte-enhancing factor 2, a transcription factor that binds to a regulatory region upstream of this cluster (Fiore et al., 2009). The transcriptional upregulation of a subset of miRNAs from the miR379-410 cluster (miR-134, -381 and -329) is necessary for activity-dependent dendritic development of rat hippocampal neurons. Furthermore, we found that the post-transcriptional regulation of the RNA-binding protein Pumilio 2 by miR-134 is essential for activity-dependent dendritogenesis. Taken together, we defined a novel MEF2-miRNA-PUM2 pathway involved in the activity-dependent regulation of dendritogenesis in primary neurons. MiR-134 localizes within dendrites of hippocampal neurons, where it can regulate the local translation of proteins important for spine structure and plasticity. However, at the beginning of this project, it was unknown how this miRNA is targeted to dendrites. I was involved in a project that aimed at identifying and characterizing the transport mechanism of miR-134 to dendrites. We found that the dendritic localization of miR-134 is mediated by the DEAH-box helicase DHX36 protein, which binds to a cis-acting element located within the loop region of the miR-134 precursor (pre-miR-134; Bicker et al., 2013). Furthermore, we showed that depletion of DHX36 increased protein levels of LIM kinase 1, a dendritically localized target of miR-134 (Schratt et al, 2006). Moreover, the depletion of DHX36 led to an increase in dendritic spine size, a similar phenotype as observed upon inhibition of miR-134 activity. In summary, we described a novel mechanism for dendritic targeting of pre-miR-134 relevant for the function of miR-134 in spine morphogenesis. Activity-dependent regulation of gene expression in the nucleus is important for the development and function of the nervous system, including synaptic plasticity and memory formation. Interestingly, several recent reports suggested that miRNAs (and/or siRNAs) might be involved in the regulation of epigenetic modifications and alternative mRNA splicing events in the nucleus of non-neuronal cells. However, whether miRNAs employ this mechanism to regulate gene expression in the neuronal nucleus was not known. A prerequisite for the study of miRNA function in the nucleus of post-mitotic neurons is the a priori knowledge of the nuclear miRNA repertoire. Therefore, using microarray and deep sequencing technologies, I identified miRNAs which are enriched in the nuclei of rat primary cortical neurons (Khudayberdiev et al. 2013; Frontiers in Mol. Neurosci, accepted for publication). Subsequently, I validated differential expression of specific nuclear-enriched miRNAs by Northern blot, quantitative real-time PCR and fluorescence in situ hybridization. By cross-comparison to published reports, I found that nuclear accumulation of miRNAs might be linked to a down-regulation of their expression during in vitro development of cortical neurons. Importantly, I found a significant overrepresentation of guanine nucleotides at the 3’ terminus of nuclear-enriched miRNA isoforms (isomiRs), suggesting the presence of neuron-specific mechanisms involved in miRNA nuclear localization. In conclusion, these results provide a starting point for future studies addressing the nuclear function of specific miRNAs and the detailed mechanisms underlying subcellular localization of miRNAs in neurons. Taken together, the results presented in my cumulative PhD thesis demonstrate that activity-dependent regulation of specific miRNAs in different subcellular neuronal compartments (dendrites, nucleus, and soma) plays an important role in neuronal morphogenesis (dendrite and spine development) and plasticity

Genome-wide microRNA screening in Nile tilapia reveals pervasive isomiRs’ transcription, sex-biased arm switching and increasing complexity of expression throughout development

MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs

MicroRNAome of Porcine Pre- and Postnatal Development

The domestic pig is of enormous agricultural significance and valuable models for many human diseases. Information concerning the pig microRNAome (miRNAome) has been long overdue and elucidation of this information will permit an atlas of microRNA (miRNA) regulation functions and networks to be constructed. Here we performed a comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained during the entire pig lifetime, from the fetal period through adulthood. The sequencing results were analyzed using mammalian miRNAs, the precursor hairpins (pre-miRNAs) and the first release of the high-coverage porcine genome assembly (Sscrofa9, April 2009) and the available expressed sequence tag (EST) sequences. Our results extend the repertoire of pig miRNAome to 867 pre-miRNAs (623 with genomic coordinates) encoding for 1,004 miRNAs, of which 777 are unique. We preformed real-time quantitative PCR (q-PCR) experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing and q-PCR data. This broad survey provides detailed information about multiple variants of mature sequences, precursors, chromosomal organization, development-specific expression, and conservation patterns. Our data mining produced a broad view of the pig miRNAome, consisting of miRNAs and isomiRs and a wealth of information of pig miRNA characteristics. These results are prelude to the advancement in pig biology as well the use of pigs as model organism for human biological and biomedical studies

Identification of the miRNAome of early mesoderm progenitor cells and cardiomyocytes derived from human pluripotent stem cells

MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.Fil: Garate, Ximena. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: la Greca, Alejandro Damián. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Neiman, Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Santín Velazque, Natalia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Luzzani, Carlos Daniel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Integrated mRNA and microRNA transcriptome sequencing characterizes sequence variants and mRNA – microRNA regulatory network in nasopharyngeal carcinoma model systems

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia among the Chinese population. Aberrant regulation of transcripts has been implicated in many types of cancers including NPC. Herein, we characterized mRNA and miRNA transcriptomes by RNA sequencing (RNASeq) of NPC model systems. Matched total mRNA and small RNA of undifferentiated Epstein-Barr virus (EBV)-positive NPC xenograft X666 and its derived cell line C666, well-differentiated NPC cell line HK1, and the immortalized nasopharyngeal epithelial cell line NP460 were sequenced by Solexa technology. We found 2812 genes and 149 miRNAs (human and EBV) to be differentially expressed in NP460, HK1, C666 and X666 with RNASeq; 533 miRNA-mRNA target pairs were inversely regulated in the three NPC cell lines compared to NP460. Integrated mRNA/miRNA expression profiling and pathway analysis show extracellular matrix organization, Beta-1 integrin cell surface interactions, and the PI3K/AKT, EGFR, ErbB, and Wnt pathways were potentially deregulated in NPC. Real-time quantitative PCR was performed on selected mRNA/miRNAs in order to validate their expression. Transcript sequence variants such as short insertions and deletions (INDEL), single nucleotide variant (SNV), and isomiRs were characterized in the NPC model systems. A novel TP53 transcript variant was identified in NP460, HK1, and C666. Detection of three previously reported novel EBV-encoded BART miRNAs and their isomiRs were also observed. Meta-analysis of a model system to a clinical system aids the choice of different cell lines in NPC studies. This comprehensive characterization of mRNA and miRNA transcriptomes in NPC cell lines and the xenograft provides insights on miRNA regulation of mRNA and valuable resources on transcript variation and regulation in NPC, which are potentially useful for mechanistic and preclinical studies. © 2014 The Authors.published_or_final_versio

Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood

<p>Abstract</p> <p>Background</p> <p>MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts.</p> <p>Results</p> <p>The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters.</p> <p>Conclusions</p> <p>We conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that post - transcriptional processes may play a significant role in regulating levels of miRNAs in tumor cells. The study also provides a customized automated computation pipeline for miRNA profiling and identification of novel miRNAs; even those that are missed out by other existing pipelines. The Computational Pipeline is available at the website: <url>http://mirna.jnu.ac.in/deep_sequencing/deep_sequencing.html</url></p

Characterization of Epstein-Barr Virus miRNAome in Nasopharyngeal Carcinoma by Deep Sequencing

Virus-encoded microRNAs (miRNAs) have been shown to regulate a variety of biological processes involved in viral infection and viral-associated pathogenesis. Epstein-Barr virus (EBV) is a herpesvirus implicated in nasopharyngeal carcinoma (NPC) and other human malignancies. EBV-encoded miRNAs were among the first group of viral miRNAs identified. To understand the roles of EBV miRNAs in the pathogenesis of NPC, we utilized deep sequencing technology to characterize the EBV miRNA transcriptome in clinical NPC tissues. We obtained more than 110,000 sequence reads in NPC samples and identified 44 EBV BART miRNAs, including four new mature miRNAs derived from previously identified BART miRNA precursor hairpins. Further analysis revealed extensive sequence variations (isomiRs) of EBV miRNAs, including terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Analysis of EBV genomic sequences indicated that the majority of EBV miRNA nucleotide variants resulted from post-transcriptional modifications. Read counts of individual EBV miRNA in NPC tissue spanned from a few reads to approximately 18,000 reads, confirming the wide expression range of EBV miRNAs. Several EBV miRNAs were expressed at levels similar to highly abundant human miRNAs. Sequence analysis revealed that most of the highly abundant EBV miRNAs share their seed sequences (nucleotides 2–7) with human miRNAs, suggesting that seed sequence content may be an important factor underlying the differential accumulation of BART miRNAs. Interestingly, many of these human miRNAs have been found to be dysregulated in human malignancies, including NPC. These observations not only provide a potential linkage between EBV miRNAs and human malignancy but also suggest a highly coordinated mechanism through which EBV miRNAs may mimic or compete with human miRNAs to affect cellular functions

Beyond the one-locus-one-miRNA paradigm: microRNA isoforms enable deeper insights into breast cancer heterogeneity.

Here we describe our study of miRNA isoforms (isomiRs) in breast cancer (BRCA) and normal breast data sets from the Cancer Genome Atlas (TCGA) repository. We report that the full isomiR profiles, from both known and novel human-specific miRNA loci, are particularly rich in information and can distinguish tumor from normal tissue much better than the archetype miRNAs. IsomiR expression is also dependent on the patient\u27s race, exemplified by miR-183-5p, several isomiRs of which are upregulated in triple negative BRCA in white but not black women. Additionally, we find that an isomiR\u27s 5\u27 endpoint and length, but not the genomic origin, are key determinants of the regulation of its expression. Overexpression of distinct miR-183-5p isomiRs in MDA-MB-231 cells followed by microarray analysis revealed that each isomiR has a distinct impact on the cellular transcriptome. Parallel integrative analysis of mRNA expression from BRCA data sets of the TCGA repository demonstrated that isomiRs can distinguish between the luminal A and luminal B subtypes and explain in more depth the molecular differences between them than the archetype molecules. In conclusion, our findings provide evidence that post-transcriptional studies of BRCA will benefit from transcending the one-locus-one-miRNA paradigm and taking into account all isoforms from each miRNA locus as well as the patient\u27s race

Small non-coding RNA landscape of extracellular vesicles from human stem cells

Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the ATMSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche.Peer reviewe