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Abstract

To enable a quick response to a variety of different stresses, cells modulate mRNA translation by repressing global translation and permitting translation of certain transcripts, thereby reprogramming the cellular proteome and adapting to the stress. In my thesis, I study two different mechanisms of how mRNA translation can be selectively regulated on subsets of mRNAs to shape the proteome.

Usually, cells recruit mRNA to a small ribosomal subunit with the use of translation initiation factor eIF4E. Cellular stresses result in the inactivation of mTORC1, which is a central controller of cellular growth and metabolism, and consequently downstream activation of a group of proteins called 4E-BPs. 4E-BPs sequester eIF4E, thus hampering eIF4E-dependent translation initiation. Although general protein production is suppressed, the translational shut-down is not complete and cells still can translate in an eIF4E-independent manner. In the first part of this thesis, I study how this eIF4E-independent translation is realised. By applying the ribosome profiling technique in cells overexpressing a constitutively-active 4E-BP1, I determined the list of transcripts resistant to eIF4E-sequestration. These transcripts have longer-than-average 5’-UTRs and rely on cap- and 5’-end dependent initiation in a condition of inactive mTORC1. I show that when 4E-BPs are active the resistant transcripts are released from eIF4E-4E-BP complex and bind via cap to eIF3D to recruit ribosomes in an eIF4E-independent manner.

In the second part of this thesis, I focus on PRRC2A/B/C proteins, which were recently linked to mRNA translation in our lab. Although PRRC2 proteins are often dysregulated in many cancers and were linked to RNA metabolism, little is known about their biological function. In our lab, we identified them as translation initiation factors. I show that the PRRC2A/B/C knock-down affects only certain transcripts, which possess upstream ORFs (uORF). The presence of uORF is sufficient to make mRNAs dependent on PRRC2A/B/C. Repression of these mRNAs suggests that lack of PRRC2s increases translation of uORFs, thereby repressing main ORF translation. Two independent mechanisms to overcome uORF repression exist – translation reinitiation and leaky scanning. I present that PRRC2A/B/C proteins promote leaky scanning on uORFs, thus permitting translation of the main ORF of uORF-containing mRNAs. This is highly relevant during cellular stress since many uORFs play regulatory roles in the stress-response.