Year: 2023
Type: dissertation
Abstract: Precise regulation of gene expression is vital for all biological processes, and \ntakes places at multiple levels including transcription, RNA metabolism, and \nposttranslational modification. In addition, mRNA molecules can be modified which \nrepresents an additional layer of gene expression regulation. The most abundant \nmRNA modification is N6-methyladenosine (m6A). This modification affects various \nbiochemical reactions including mRNA decay and translation. Moreover, m6A is \nimplicated in a wide range of physiological processes such as differentiation and \ngametogenesis, and dysregulation of the m6A modification has been associated with \nmultiple types of cancer and other diseases. The m6A mark is deposited on mRNA \ntranscripts by methyltransferase complexes known as m6A writers. While the \nbiochemical reaction to form m6A-modified RNAs is well characterized in vitro, m6A \ndeposition in vivo remains poorly understood. \nHere, I used the budding yeast Saccharomyces cerevisiae to dissect how \nm6A deposition is regulated. In budding yeast, m6A is only deposited during early \nmeiosis. Using a multifaceted approach, including proteomics, yeast genetics, \ngenomics, and a new method to rapidly determine relative m6A levels across mRNA \nsamples (m6A-ELISA), I identified and characterized the yeast m6A writer complex. \nI found that the yeast m6A writer consists of six proteins (Ime4, Mum2, Slz1, Kar4, \nYGL036W, Dyn2), of which 5 are essential for m6A deposition (Ime4, Mum2, Slz1, \nKar4, YGL036W), and of which three were previously known (Ime4, Mum2, Slz1). \nSequence and structure prediction data reveal that the m6A writer is highly similar \nbetween yeast, plants, and mammals. Apart from m6A deposition, several of the \nwriter components have additional cellular functions: Kar4 has a separate function in \nmating, and a subcomplex (Ime4, Mum2, YGL036W, and Kar4) promotes meiosis in \nan m6A-independent manner. My work forms a starting point for understanding the \nmolecular basis for m6A deposition as well as new molecular functions of m6A writer \ncomplexes.
Author: Ensinck, Imke Maria Franciscus
Language: English
Topic: RNA modifications and cancer
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6990588819