Exploring the functional and structural role of Paxbp1

Abstract

Pax7 is widely recognized as a prominent marker for muscle satellite cells (MuSCs). Pax3/Pax7 binding protein 1 (Paxbp1) was initially identified as a Pax7 and Pax3-interacting nuclear protein in our laboratory by yeast two-hybrid (Y2H) screening. Our investigation revealed that Paxbp1 exerts control over a critical checkpoint during early activation of muscle satellite cells. Notably, the loss of Paxbp1 results in a failure of muscle satellite cells to undergo full activation and to re-enter the cell cycle. To gain further insights into the Paxbp1 functions, we conducted Bio-ID screening in C2C12 myoblasts stably expressing Paxbp1. Surprisingly, we observed a marked enrichment of spliceosome proteins, with Tfip11 being particularly prominent. Tfip11 has previously been implicated in the disassembly of intronlariat spliceosomes (ILS). Furthermore, RNA-Seq analysis revealed high similarities in splicing defects between Paxbp1-knockout and Tfip11-knockout muscle satellite cells, suggesting that Paxbp1 may function as a spliceosome protein primarily involved in the disassembly of ILS. However, the impact of Paxbp1 gene knockout differed between MuSCs and myoblasts. While Paxbp1 knockout in quiescent MuSCs resulted in severe phenotypic consequences including inability to re-enter the cell cycle and subsequent apoptosis, Paxbp1 knockout in myoblasts only led to minor defects. Specifically, Paxbp1-deficient myoblasts could still proliferate and differentiate without showing signs of apoptosis as observed in MuSCs. In the mouse genome, Gcfc2 is the sole homologue of Paxbp1, sharing a high sequence similarity. Gcfc2 has also been implicated in RNA splicing. Additionally, isothermal titration calorimetry (ITC) experiments revealed that Paxbp1 and Gcfc2 exhibit mutually exclusive binding with TFIP11. These results could explain why loss of Paxbp1 in myoblasts only has minor effects. However, Gcfc2 was unable to compensate for the loss of Paxbp1 in MuSCs. To further understand the roles of Paxbp1 and Gcfc2 in RNA splicing, I knocked in a Flag tag separately at the N-terminus of Paxbp1 and Gcfc2 in HeLa cells by CRISPR/Cas9. I then immunoprecipitated Flag-Paxbp1 or Flag-Gcfc2 and subjected the eluates by the Flag peptide to mass spectrometry. Preliminary mass spectrometry results indicated that both Paxbp1 and Gcfc2 are associated with a common subset of proteins involved in RNA splicing. Additionally, both Paxbp1 and Gcfc2 also bind a distinct subset of proteins. Moreover, we showed that the levels of Myc protein (low in myoblasts and high in MuSCs) could underlie differential responses to loss of Paxbp1 between myoblasts and MuSCs. Overexpression of c-Myc in Paxbp1-deficient myoblasts resulted in a reduced proliferation rate and more apoptosis. Furthermore, to gain comprehensive understanding of Paxbp1, it is crucial to determine its three-dimensional structure together with Tfip11. By co-expressing truncated Paxbp1 with Tfip11, we successfully purified a Paxbp1-TFIP11 complex using an E. coli expression system. Preliminary data from cryo-electron microscopy (Cryo-EM) studies, combined with Alphafold2 predictions, suggest that these two proteins adopt a linear shape. In conclusion, my work on Paxbp1 has provided valuable insights into the mechanism by which Paxbp1 exerts its cellular functions in myoblasts and MuSCs.

Date of Award	2024
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Zhenguo WU (Supervisor)