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Non-coding RNAs and their role in developmental networks

Final Report Summary - NCRNASDEVNET (Non-coding RNAs and their role in developmental networks)

Non-coding RNAs and their role in developmental networks

One of the most intriguing questions in Biology is how the complex body plans of multicellular animals (metazoans) unfold from a single fertilized egg. What we know so far is that complex regulatory networks, formed mainly by the interaction of transcription factors (proteins that regulate transcription) and their target sequences in the DNA.
Before the genome-sequencing era, it was assumed that increasing morphological complexity should correlate with an increase in the complexity of the protein repertoire; however it is now apparent that the number of protein-coding genes shows little variation across the metazoans. In contrast, the extent of non-coding sequences (genome sequences that will not be translated into protein) is highly correlated with developmental complexity. This includes the regulatory modules bound by transcription factors but also numerous non-coding RNAs (ncRNAs) genes with regulatory capabilities. Many of these ncRNAs have proven roles in development and show exquisite regulation, both in cell type and developmental stage. Their functions include all different aspects of gene expression, from chromatin structure and transcription to translation and protein stability.
In spite of the importance of non-coding region shown by comparative genomics and the support for roles of ncRNAs in development-associated gene regulation, no systematic analysis of genome-wide ncRNA expression and participation in development of an embryonic tissue has been conducted to date. Systematic long non-coding RNA analysis has mostly been conducted either in cultured cell models of differentiation, a system that lack the exquisite temporal and spatial regulatory requirements needed for precise and complex control in vivo, or in whole embryo tissue, which precludes the identification of ncRNAs expressed in very specific cell types or stages.

Our main objective is to conduct a detailed analysis of ncRNA expression and assessment of their role within the developmental regulatory networks, using mesoderm development in the fruit fly Drosophila melanogaster as a very well established model system. The mesoderm is a tissue layer in the early embryo that will ultimately form a variety of internal tissues, such as the different types of muscles, the heart, the blood (or hemolymph in insects), etc. We take advantage of the accumulated knowledge on the regulatory network that controls early mesoderm development to integrate the information we found regarding ncRNAs in a meaningful biological context.
We performed deep sequence of the total transcriptome of mesodermal as well as whole-embryo cells, at three different time intervals during early mesoderm development, when the fate of the different cells is being determined. The use of tissue-specific samples in short time intervals increase our sensitivity to reliable find new ncRNAs which are difficult to annotate because low expression. We increased our sensitivity by also sequencing nuclear RNA from mesodermal cells, since we expect that molecules with a role on transcriptional regulation to be located in the nucleus. We also used complementary approaches (such as sequencing the transcript ends and using chromatin marks information) to precisely define the start of the transcripts, which allows us to understand the biogenesis of the different classes of ncRNAs.
In a collaborative effort with Anton Enright lab (EMBL-EBI), we created a set of newly discovered ncRNAs, which includes around 140 genes in its most conservative version. Even with a limited number of different biological conditions, most of these genes are found differentially expressed across tissue or time, confirming the highly regulated nature of ncRNAs. We detected different classes of ncRNAs, including long intergenic ncRNAs, stable RNAs originated from enhancers, stable transcript originating divergently from promoters of other genes, antisense RNAs and antisense promoter-overlapping ncRNAs.
We also obtained a similar set of previously annotated ncRNAs that are confidently expressed at these time stages, and integrate the two sets for functional studies.
Once the transcripts are annotated, it is important to further characterize their expression in the context of the embryo. We detect a number of these transcripts by using fluorescent antisense probes, which not only validates these new ncRNAs, but also helps to assign possible function to the genes. For example, one new transcript is expressed in very early embryos in a pattern typical of the genes that establish the antero-posterior polarity in the embryo.
The most definitive evidence of a role of these ncRNAs in development will be of course to delete them or inhibit their expression in actual flies, and them watch and characterize the development of the embryos in these fly lines. The recently developed CRISPR/Cas9 genome-editing technology is being used to generate functional knockouts of a selected set of candidates. In addition to probe functionality, these deletions will be a valuable tool for investigating the molecular phenotype of selected candidates, as well as their role in the developmental gene-regulatory networks, by studying how their absence impacts the expression of other genes and the function of nearby enhancers.

Understanding the regulatory networks behind different developmental processes is not only a relevant goal for basic biology, but also an essential step for the study, diagnosis and treatment of developmental diseases. All evidence points to the fact that ncRNAs participate in developmental regulatory networks, making it necessary to thoroughly assess their role and integrate them in the transcription factor networks already constructed. Given our very limited knowledge of the functions of ncRNAs, to perform this in complex organism like mammals would be gigantic task, considering that the number of non-coding sequences increase considerably with organismal complexity. Therefore, to understand the role of ncRNAs will be much more tractable in a simpler model organism, such as the fly. In particular, the tissue-specific nature and temporal dynamics of the data obtained in the context of the developing embryo offers biological insights that no tissue culture system can provide.
The first results from our project shows that an integrative approach to the study of development in vivo within a simple organism can lead to the discovery of new ncRNAs and transcript classes associated with different developmental systems. We expect that the finalization of the functional and molecular studies will provide very important insight into the role that these classes of transcripts play in the complex regulatory networks that drive development and this, in turn, will increase our understanding of how they are organized and how they can be affected during disease.