Genomic engineering to decipher the proteome responsible for CUT60 termination hos Institut for Plante- og Miljøvidenskab - Københavns Universitet

Most DNA of eukaryotes does not code for proteins. However, RNA polymerase II (responsible for transcribing coding sequences) is also responsible for transcribing non-coding sequences into long non-coding RNA (lncRNA). Knowledge about the regulatory function of lncRNA transcription is still limited. One lncRNA that has been studied in budding yeast, CUT60, is an example of the importance of the regulation of this type of transcript in the rest of the genome. CUT60 is located in tandem with a downstream protein-coding gene, ATP16, that encodes a subunit of the ATP synthase and plays an important role in maintaining the mitochondrial DNA. The efficient termination of CUT60 prevents transcriptional interference of CUT60 over ATP16, thus leading to a proper ATP16 expression and maintaining mitochondria integrity. This project focuses on investigating what are the factors that allow this efficient termination of CUT60 transcription.

To resolve the factors associated with the efficient termination of CUT60 there are available classic proteomics approaches that rely on mass spectrometry, but they are challenging. Instead, this project uses an emerging and powerful method, called Epi-Decoder, that relies on DNA barcoding and next-generation sequencing to decode the proteins that interact in a single locus. In this method, a yeast library is generated by combining other two libraries: one where each protein of the genome carries an epitope tag and the other with a unique barcode at the locus of interest, CUT60 (generated by CRISPR-Cas9 technologies). Followed by chromatin-immunoprecipitation against the common tag, the barcodes can be recovered, amplified, and high-throughput sequenced to decode the local proteome composition.

In summary, this project focuses on the application of Epi-Decoder to decipher the chromatin proteome responsible for CUT60 termination in budding yeast, and thus uncover the regulatory function of this transcript unit.

 

Methods used: CRISPR-Cas9 genomic engineering, synthetic genetic array, robotics, high-throughput sequencing, S. cerevisiae essential protocols

 

If you are interested in this project or would like to know more about it, please contact Desiré García Pichardo (dgp@plen.ku.dk) or Sebastian Marquardt (sebastian.marquardt@plen.ku.dk).

 

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