Divergence of the RNA Recognition Motif in Vertebrate LARP6 Proteins
The La-related proteins (LARPs) share a conserved RNA-binding unit composed of a La Motif and an RNA Recognition Motif (RRM), which recognizes diverse RNA substrates. LARPs use different surfaces on their RRMs to bind their respective RNA ligands. LARP6, the least understood LARP subfamily, binds to the stem loop (SL) structure found in the 5’ untranslated region of collagen types I and III. Previous work showed that the exchange of the LARP6 RRM for the RRM from Genuine La (LARP3) protein disrupts RNA binding activity. Across the vertebrate LARP6 proteins, there are regions of localized divergence within the RRMs, raising the question whether LARP6 RRMs are exchangeable. To understand the role of RRM sequence divergence in the structure and function of LARP6, chimeric proteins were constructed in which the RRMs are exchanged between human and fish species. The chimeric proteins were cloned as three constructs: full-length proteins, C-terminal deletions (∆CTD), and isolated “La Modules”. The chimeras were recombinantly expressed in E. coli with N-terminal histidine tags for detection and affinity purification. The full-length and ∆CTD constructs of the human/fish chimeras (consisting of human sequence flanking a fish RRM) required a solubility tag for recombinant expression. In contrast, the human/fish chimeric La Module constructs did not. This result suggests that the N-terminal region may form species-specific interactions with the RRM, that are disrupted by the chimera. The chimeric La Modules were recombinantly expressed and purified. They were characterized for protein stability via limited trypsinolysis assays and RNA binding activity with electrophoretic mobility shift assays. As predicted, the stability and RNA binding behavior of the chimeras appears to depend on the RRM. Specifically, the chimeras composed of human La Motif and fish RRM are more similar to the native fish La Modules than the native human La Module. This work suggests the sequence divergence within the RRM defines the structural stability and RNA binding behavior of the La Module.
Carrizales, M. (2019). <i>Divergence of the RNA recognition motif in vertebrate LARP6 proteins</i> (Unpublished thesis). Texas State University, San Marcos, Texas.