Investigations of the molecular interactions of two RNA-binding proteins, LARP4A and LARP4B

Student thesis: Doctoral ThesisDoctor of Philosophy


RNA-binding proteins (RBPs) bind to RNA targets and control a myriad of cellular functions such as transcription, mRNA localisation, stability, polyadenylation, splicing and decay. Mis-regulation of these processes are known to be prevalent in diseases such as cancer or neurodegeneration.

This project aims to characterise two RNA-binding proteins, namely LARP4A and LARP4B from the La-related RNA-binding protein (LARP) family. LARPs are a group of evolutionary conserved RBPs that regulate RNA metabolism identified by the presence of a conserved region termed the La-module which modulates the interaction to RNA. LARP4A and LARP4B have high amino acid similarity, share the same protein partners, but have different target RNA. Elucidation of the exact binding mechanism and their different RNA preference are two key areas of focus in my research.

LARP4A is known to interact with polyA sequences located in the 3’ ends of mRNAs. LARP4B targets AU-rich stretches, typically found in the 3' untranslated region (UTR) of many mRNAs. Both proteins are known to control key cellular processes such as mRNA stabilisation and translation, they localise to stress granules and have been recently linked to cancer. In addition to their interaction with RNA they are both known to interact with PolyA binding protein (PABPC1) which is a well characterised protein that is involved in the regulation of RNA metabolism, polyA lengthening and the termination of translation. The interaction to PABPC1 involves a short peptide sequence in the N-terminal regions of LARP4A and LARP4B, termed PAM2w. Although not well characterised, evidence of a second interacting region named PABP-binding motif (PBM) also exists downstream of the La-module.

In my project, a range of deletion and point mutants of LARP4A and LARP4B proteins were designed and generated from recombinantly proteins expressed in Escherichia coli to be tested for their properties. Biochemical assays such as Electrophoretic Mobility Shift Assays (EMSA) and Microscale Thermophoresis (MST) were employed to characterize the binding affinity and specificity between different protein constructs and RNA. Furthermore, circular dichroism (CD) was used to analyse any changes of secondary structure from mutant to mutant. 

Previous work from our lab shows that LARP4A recognises polyA RNA via a binding mechanism on the N-terminus mediated by disordered regions involving a protein- binding motif PAM2w, rather than the usual RNA-binding La-module. Furthermore, I tested binding to oligoA RNA using PAM2w motif alone and showed that a contiguous region at the N-terminus of LARP4A is required for oligoA RNA binding, and regions only encompassing the PAM2w motif alone are not enough to bind RNA. For LARP4B, to characterise its RNA recognition I investigated the N-terminal domain (NTD) that comprises the La-module and an N-terminal region, by systematically trimming the N- terminal region and have obtained deletion mutants, results show a gradual decrease in binding affinity towards AU-rich RNA the more N-terminal residues removed. Within the N-terminal region there also seems to be an unknown flexible RNA-binding region which is responsible to bind to RNA. The La-module of LARP4B shows increased binding affinity to AU-rich RNA when compared to the extremely weak binding of LARP4A to oligoA RNA. Within the La-module there are 6 conserved residues known to be vital for RNA-recognition. Using sequence alignment, we have generated point mutants in LARP4B that revealed little to no difference in RNA binding using our assays, suggesting that these amino acids play a minor role in RNA-recognition in LARP4B. 
Date of Award1 May 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMatthias Krause (Supervisor) & Maria Conte (Supervisor)

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