The minority of eukaryotic DNA codes for proteins. Despite this, non-coding DNA regions are also actively transcribed by RNA polymerase II into long non-coding RNAs (lncRNA). Knowledge about the regulatory function of lncRNA transcripts is still limited. Part of these transcripts forms protein-RNA complexes to modulate different activities, such as epigenetic regulation or nuclear organization. However, screening which proteins are bound to a specific RNA transcript (coding or lncRNA) is challenging.
To solve which proteins are bound to a specific RNA transcript, we are going to skip classic approaches that rely on mass spectrometry, and therefore, avoid the challenges that it implies. Instead, we are going to develop a completely new technique, which we call RNA-decoder. RNA-decoder relies on DNA barcoding and RIP(RNA immunoprecipitation)-sequencing to decipher the proteins that bind in an RNA transcript. In this method, we are going to use a yeast library that has already been generated in our lab where each strain combines one different protein marked with a tag, with two unique barcodes inserted in its genome, one within a coding region, and the other at a non-coding region. After pooling the entire library, we can perform a RIP-sequencing protocol, where we can immunoprecipitate all yeast proteins using the common tag. Followed by RNA purification, specific cDNA synthesis and PCR-amplified libraries; the unique barcodes can be high-throughput sequenced. Subsequent bioinformatics analyses will reveal the specific proteins associated with the different RNA transcripts in this model, one mRNA and one lncRNA.
In summary, this project focuses on the development of a promising new method to screen novel RNA-protein complexes. Through its application, we will be able to uncover the functional differences between RNA-protein complexes in a coding and a lncRNA transcript.
Methods used: RIP-seq, high-throughput sequencing, S. cerevisiae essential protocols
Keywords (5): RNA-decoder, RIP-seq, RNA-protein complexes, CRISPR-based genomic engineering, DNA barcoding, lncRNA