Abstract:
The temporal and spatial expression of genes is required for maintaining cellular asymmetry, proper embryonic development, neuronal function, and cell fate. In mouse embryonic fibroblasts (MEFs) this cellular asymmetry is generated by localizing various cellular mRNAs to the protrusions (lamellipodia/filopodia). Among those mRNAs, β- actin mRNA plays a major role in defining cellular asymmetry by its localization to the cell periphery. Upon mRNA localization and translation, β-actin protein helps the cells to respond to extracellular cues and to move during extracellular matrix remodeling to maintain tissue homeostasis and tissue repair, traversing changes in local tissue environments as needed in tissue degradation, repair or regeneration. Under normal trophic conditions, the localization of β-actin mRNA to the cellular protrusions of fibroblasts or growth cones in neurons is regulated by a cis-acting localization element or localization signal known as zipcode (in case of β-actin, this is a 54 nt long sequence in the 3’UTR of the β-actin mRNA adjacent to its stop codon) together with trans-acting factors, mainly RNA-binding proteins (RBPs) that either bind directly to the zipcode or regulate the binding of other RBPs to it. In the case of the motor-driven movement of these localized mRNAs, such RNA-protein complexes are then tethered to molecular motors such as kinesin, dynein, or myosin, to form transport or locasome complexes. Thus, messenger ribonucleoprotein particles (mRNPs) that act as functional units not only contain the information for an encoded polypeptide but also determine the precise spatio-temporal regulation of its translation, thereby facilitating the correct subcellular localization of the translation product. It has been shown that the localization of β-actin mRNA is dependent on the binding of the zipcode-binding protein ZBP1 (an RBP of the conserved VICKZ RNA-binding protein family) to its cognate site present in the 3’UTR of the mRNA. ZBP1 (also called IGF2BP1 or IMP1) interacts with the zipcode via two K-homology (KH) RNA-binding domains by RNA looping mechanism and is required for β-actin mRNA localization in migrating cells including fibroblasts and neurons. In addition, in fibroblasts, it is also known that it controls the translation of β-actin by blocking the assembly of ribosomes at the start codon. Apart from ZBP1, the RBPs IGF2BP2, RACK, KHDRBS1/Sam68, and FMR1 play important roles during the localization of the mRNA. To obtain a complete picture of the associated proteome of any mRNA has been challenging. The high throughput methods available so far (like CLIP, MS2 pull down) mainly fail in the identification of indirect or transient interactors of specific RNAs. To solve these issues, I applied a BioID method where a protein of interest is fused to a mutant version of the E.coli biotin ligase BirA (BirA*), which biotinylates accessible lysine residues of proteins present in its vicinity. After cell lysis, biotinylated proteins can be isolated by streptavidin affinity purification and identified using standard mass spectrometry techniques. In this thesis, I report that tethering of BirA* to a specific localized, MS2- tagged mRNA does not only allow the identification of its associated proteins but can also be used to probe the environment of this mRNA. This approach allows, with high confidence, to identify novel functional β-actin interactors like FUBP3/MARTA2, STAU1, and STAU2. FUBP3 is an RBP from the conserved FUBP family of proteins. FUBP3 shown to mediate the dendritic targeting of MAP2 mRNA in neurons. In this thesis, I report FUBP3 to bind to and facilitate localization of β-actin mRNA to fibroblast protrusions. By immunoprecipitation and in vitro binding assays, I could demonstrate that it binds 460 nt downstream of the stop codon in the β-actin 3’ UTR and participates in the localization of the mRNA to the cellular protrusions. Apart from BirA* I also applied direct MS2-MCP pull-down APEX2-mediated biotin labeling of beta-actin associated proteins and compared the obtained datasets of the proteins that bind to the β-actin mRNA directly or via transient interactions. The established method convincingly shows 1. Additional proteins which could be a part of the β-actin localization complex. Amongst all these proteins, FUBP3 has shown to be a part of the β-actin localization complex for the first time. 2. FUBP3 to bind to downstream of the localization element at the 3’UTR of β-actin mRNA and is essential for the localization of β-actin mRNAs at the protrusions of fibroblasts. 3. Comparison of the β-actin proteome under serum-starved and unstarved conditions and the difference between the associated RNA interacting proteome under these two conditions.
proximity biotinylation
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