Divergence of the RNA Recognition Motif in Vertebrate LARP6 Proteins
Date
2019-05
Authors
Carrizales, Melissa
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Abstract
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.
Description
Keywords
LARP6, RRM
Citation
Carrizales, M. (2019). <i>Divergence of the RNA recognition motif in vertebrate LARP6 proteins</i> (Unpublished thesis). Texas State University, San Marcos, Texas.