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TRANSLATE Report Summary

Project ID: 617837
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Mid-Term Report Summary - TRANSLATE (Specificity of translational control during unfolded protein response)

We have completed the first phase of the project to identify and analyse functionally important secondary RNA structures in the 3’ UTRs of mRNAs. We developed a new methodology called hiCLIP (hybridization iCLIP) that identifies RNA duplexes bound by double-strand RNA-binding proteins (dsRBPs). This allowed us to examine interactions between proteins and double-stranded RNAs in human cells, and studied the function of identified structural motifs in RNAs in the stability of mRNAs and translation of proteins.

We have completed the first phase of analysing functionally important RNA-RNA interactions in HEK293 cells and studied their impact on mRNA translation and stability. We used the dual luciferase translation assay system to validate the functional relevance of intra-mRNA interactions that involve long-range contacts between two distant regions of the XBP1 3´ UTR. The 3´ UTR of this mRNAs was added to the luciferase coding sequence, which enabled us to test the role of dsRNA regions with appropriate mutations. We tested the function of dsRNA by first disrupting the RNA-RNA interaction with mutations, and then rescuing it with compensatory mutations in the complementary strand. Part of this work has been reported in the publication ‘hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1’.

We developed a computational pipeline of regulatory dsRNA elements and RNA structures and their positional principles. We made progress with studies to understand how assessed how the activity of RBPs depends on their binding position relative to the stop codon, poly-A site, or binding sites of other RBPs or microRNAs. We found that STAU1 recognises multiple RNA motifs that are clustered on the 3’ UTR, and we currently study how the spacing between of these RNA motifs, and the relative arrangement of sequence and structural elements within mRNAs, affects mRNA stability. We test these positional strategies by placing the RNA motifs at different positions within the 3´ UTR of the luciferase reporter, or by adding combinations of multiple RNA motifs that will be positioned at variable spacing relative to each other.

We also developed new computational methods to analyse high-throughput data of protein-RNA interactions defined by iCLIP and related methods (Haberman et al, Genome biology, in press), and study how genomic variation at the sites of these interactions affects the quality control and evolution of alternative splicing (Attig et al, eLife, 2016).

Finally, we developed machine learning methods to count different types of cells and analyse their morphology in high-resolution images of human brain sections (Soreq et al, Cell Reports, in press). This allows us to define the earliest markers of age-related changes in the brain, so we can then further pursue the changes in UPR, RNA metabolism and mRNA translation that are relevant to aging and age-related neurodegenerative diseases.

Reported by

United Kingdom
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