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Role of cystines in the structure and function of the RNA‐binding protein LARP6

La‐related protein 6, formerly known as Acheron, is an RNA binding protein associated with the translation of mRNAs that encode type‐I collagen. This protein belongs to a superfamily of RNA‐binding proteins known as the La‐related proteins, which share a conserved La Module consisting of a La‐Motif (LaM) and RNA Recognition Motif (RRM). Although extensive research on the mechanism of binding by LARP6 is being performed, very little is known about the physiological conditions that induce the protein to bind to its ligand. A multiple sequence alignment of the LARP6 proteins from vertebrates identified a fully conserved cysteine near the RRM (position C258 in the human protein), and two semi‐conserved cysteines near the C‐terminal region (positions C378 and C490 in human LARP6). To determine if disulfide bonds were present in the protein, the reducing agent dithiothreitol (DTT), was added during purification. In the presence of DTT, LARP6 eluted from a size exclusion column at a volume that is indicative of an increase in the hydrodynamic radius of the protein. Therefore, we hypothesize that at least one pair of the conserved cysteine residues is involved in intramolecular disulfide bonding. Using the human LARP6 (HsLARP6) as a model, the location of the structurally relevant disulfide bonds is being identified using a combination of site‐directed mutagenesis, chemical modification, and mass spectrometry. By mutating each cysteine to serine, we can monitor changes in protein stability through limited proteolysis and chemical and thermal denaturation assays. Additionally, the effects of cysteine mutagenesis on the biochemical activity of LARP6 will be assessed using electrophoretic mobility shift assays against the previously identified stem‐loop structure in the 5′UTR of the COL1a1 mRNA. The identification of the existence and function of disulfide bonds within LARP6 will provide further understanding of the physiological function of this posttranscriptional regulator.