L1-DIGEORGESYNDROME · Role of LINE-1 retrotransposons in the human disease DiGeorge Syndrome

Role of LINE-1 retrotransposons in the human disease DiGeorge Syndrome

A large fraction of mammalian genomes is comprised of Transposable Elements, which are pieces of mobile DNA. Indeed, retrotransposon derived-sequences comprise roughly 50% of the mammalian genome and their activity has extensively shaped our genome during evolution. Currently, a small fraction of non-LTR retrotransposons, termed LINE-1s and SINEs, is active in the human genome (RetroComentent (RC) L1s, Alus and SVAs). These elements move in our genome using an intermediate RNA and a reverse transcriptase activity by a copy and paste mechanism. Their ongoing and random mobilization can impact the human genome, leading to the appearance of a wide range of human genetic disorders. Due to their mutagenic potential, the host tightly controls the activity of retrotransposons. However, how the cell controls the activity of these elements is not fully understood. During the course of this project, we have confirmed our previous observations showing that the Microprocessor (Drosha-DGCR8 complex) controls the activity of mammalian retrotransposons. Furthermore, we have shed light on the molecular mechanisms underlying this regulation. Indeed, we have demonstrated that the Microprocessor can process the 5´UTR of RC-L1s, which presumably adopt a strong and stable RNA secondary structure in vitro and probably in vivo. Similarly, we have shown that RNAs derived from RC-Alus are also a substrate for the Microprocessor complex. Thus, these data suggest that the Microprocessor represses mammalian retrotransposons by directly binding and processing secondary RNA structures contained in their intermediate RNA molecules. These results were published in Nature Structure and Molecular Biology and the CIG fellow is the first author: Heras SR, et al. The Microprocessor controls the activity of mammalian retrotransposons. Nat Struct Mol Biol. 2013 Oct;20(10):1173-81. Some of the authors of this study were invited to write a commentary for the journal Mobile Genetic Elements, and the CIG fellow was first and co-corresponding author in this article: Heras SR. et al. Control of mammalian retrotransposons by cellular RNA processing activities. Mob Genet Elements. 2014 Mar 6;4:e28439. As follow up of the project, and considering that the synthesis of microRNAs (miRNAs) is the best-known function of the Microprocessor, we next tested whether miRNAs generated by the Microprocessor could also control L1 activity. Interestingly, we have demonstrated that the well-known microRNA let-7 directly binds to a coding region of the L1 mRNA, reducing the translation of the L1-encoded proteins. Specifically, our data suggest that let7 reduce the translation of the second open reading frame (ORF2) contained within the L1 mRNA, and is remarkable that ORF2 codes for the enzymatic activities required for LINE-1 retrotransposition. Additionally, we used a cell culture-based L1 mobilization assay and demonstrated that let-7 reduces LINE-1 retrotransposition in vivo. Altogether, these data suggest that let-7 reduces mammalian L1 retrotransposition by interfering with ORF2p translation. The let-7 family of miRNAs is known to have relevant roles in development, differentiation and proliferation. Thus, in this study we have described a new role for let-7 in controlling the activity of active mammalian LINE-1 retrotransposons, which could help to maintain genomic integrity over evolution. We hypothesize that this mechanism can be, at least to some degree, responsible for some of the functions ascribed to the let-7 family of miRNAs. On the other hand, a common human genetic disorder known as DiGeorge Syndrome or 22q11.2 deletion Syndrome (22q11 DS) is characterized by the deletion of a fragment of chromosome 22 during meiosis that includes the DGCR8 gene. 22q11 DS affects 1/4000 newborns, being the most common microdeletion in humans. 22q.11 DS patients suffer from cardiac malformations, schizophrenia, and immunological problems among others. During the project, we edited the genome of human embryonic stem cells (hESCs) to generate homozygous and heterozygous DGCR8 knockout cells, using CRISPR/Cas9 methodology. Importantly, hESCs can be differentiated to neuronal Progenitor Cells (NPCs) and NPCs can be differentiated further into mature neurons. Thus, we have established an early human embryo model to determine the role of DGCR8 in the human 22q11.2 deletion syndrome. Additionally, the CIG-fellow is a co-author in a recent study that demonstrated that LINEs are active in cultured NPCs and mature neurons (Macia A. et al. Engineered LINE-1 retrotransposition in non-dividing human neurons. Genome Res. 2016 Dec 13. pii: gr.206805.116. [Epub ahead of print]). In sum, the 22q11.2DS model generated will allow us to clarify the molecular mechanisms underlying impaired neurogenesis associated to DGCR8 depletion, including the impact derived by an exacerbated L1 retrotransposition activity in NPCs and mature neurons.

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