Uncovering the mysterious function of the most deeply conserved non-coding RNA sequences in insect genomes

One of the major goals of evolutionary genomics is to identify those regions that are functionally important and are maintained by natural selection. In contrast to protein-coding regions that are relatively easy to identify (e.g. by looking for open-reading frames), other functional elements such as transcriptional and translational regulatory regions may be short and lacking a clear sequence motif that would allow them to be detected in a single genome. A recent collaborative study of the Tauber (Leicester) and Ott (Warwick) research teams, we compared the genome sequence of insect species and searched for highly conserved sequences as more important sequence elements are more likely to be conserved over time than less important sequence elements, leaving behind a ‘footprint’ of functionality. Our study [1] has discovered intriguing conserved regions in some 5′ UTRs in a large set of genomes. Features, conservation patterns, and the age of these regions compel us to think that these must have a fundamentally important role, likely to do with translation. Two of the sequences that we have identified are oldest and most ubiquitously conserved non-coding elements yet described in the animal kingdom. These ancient conserved non-coding elements are associated with the two ribosomal stalk components and would have been functional in some of the earliest animals.
We would now like to carry experimental work that would uncover the functional role of these conserved non-coding sequences (CNS). In one part of the project we will try to identify the proteins that bind to these CNS. Here, we will use a recently published technique [2], called interactome capture, for identifying the active RNA binding proteins (RBP). Briefly, polyadenylated synthetic RNAs carrying the target sequence would be used and will be cross-linked to the RBPs by UV. The covalently bound proteins will be captured with oligo(dT) magnetic beads, and after stringent washes, the mRNA interactome will be determined by quantitative mass spectrometry (MS). The candidate RBP that would be identified will be further analysed, by expression and miss-expression analysis (e.g. RNAi knockdown) in Drosophila or other insect model system.
In the second part of the project, you will test the function of these sequences by deleting these CNS using transgenic techniques in Drosophila. You will also carry expression analysis of different tissues and different developmental times in various insects. This information will allow us the specific and localised miss-expression of the target sequences and the RNA binding protein, and will also shed more light on the function of these sequences.

[1] Harmston N, Barešić A, and Lenhard B. 2013. The mystery of extreme non- coding conservation. Phil. Trans. R. Soc. B. 368: 1471-2970; doi:10.1098/rstb.2013.0021 1471-2970

[2] Davies N, Krusche P. Tauber E and Ott S. Analysis of 5’ untranslated regions reveals extraordinary conservation of novel non-coding sequences across wide range of Arthropods. Submitted to BMC Evolutionary Biology.

[3] Castello A, Horos R, Strein C, Fischer B, Eichelbaum K, Steinmetz LM, Krijgsveld J, Hentze MW. 2013. System-wide identification of RNA-binding proteins by interactome capture. Nature Protocols. 8(3):491-500. doi: 10.1038/nprot.2013.020