טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZach Lolita
SubjectCellular Adaptations to Protein Misfolding: AIRAP
Translastion is Regulated by Means of Leaky
Scanning
DepartmentDepartment of Medicine
Supervisor Dr. Ariel Stanhill
Full Thesis textFull thesis text - English Version


Abstract

Initial steps in protein synthesis are highly regulated processes as they define the reading frame of translation and commit the translation machinery to begin the elongation phase. Eukaryotic translation initiation is a process facilitated by numerous factors, eukaryotic initiation factors (eIFs), aimed to form a “scanning” mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs). uORFs are prevalent cis-regulatory sequence elements in the transcript leader sequences of eukaryotic mRNAs. The majority of uORFs is considered to repress downstream translation of the main ORF by the consumption of functional pre-initiation complexes (PIC). This mode of regulation is called "re-initiation" and is governed by the phosphorylation status of the α subunit of eIF2. Phosphorylation of eIF2α is controlled by various cellular stresses and is activated by various kinases.

Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied, much less under stress conditions. Leaky scanning is a mechanism that allows for the 40S ribosomal subunit to bypass the initial AUG start codon and begin translation at further downstream AUG start codons. Several factors affect initiation codon selection and leaky scanning: elements in cis include the context surrounding the AUG codon (known as Kozak context) and near cognate AUGs; elements in trans include eIF1, which is a key factor in discriminating against initiation at AUG codons that have a weak nucleotide context.

In this thesis we have identified arsenite-inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions, in a leaky scanning regulatory manner. AIRAP transcript contains a single uORF in a poor-Kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF Kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor Kozak context of a single uORF transcript.