Deciphering Structural Stabilities of Pre-messenger RNA Splicing Protein Dib1 Paralogs from Cyanidioschyzon merolae

Pre-messenger RNA (pre-mRNA) splicing is facilitated by the spliceosome, a dynamic, multimegadalton ribonucleoprotein complex that catalyzes the removal of intronic sequences from pre-mRNA. Dib1 is a small, essential U5-associated protein located in the catalytic core of the canonical precatalytic spliceosome. Departure of Dib1 from the spliceosome is necessary for the transition to a catalytically active spliceosome. Dib1 is highly conserved, including its presence as one of only ∼50 core splicing proteins in the extremophile red alga Cyanidioschyzon merolae. Furthermore, two paralogs of Dib1 have been identified in the C. merolae genome. To understand the nature of the two C. merolae Dib1 paralogs, this study presents their biochemical characterization. Here, we determine the free energy of unfolding of both C. merolae Dib1 proteins, finding that despite sharing ∼97% sequence identity, the stabilities of the two paralogs differ substantially. Additionally, only one C. merolae Dib1 paralog displayed a pH-dependent stabilization. Through mutational analysis, we identified two critical residues involved in the stability difference between these paralogs and propose a structural explanation for the observed difference. The collective differences in stability and pH dependence of the two C. merolae Dib1 paralogs suggest that the paralogs may play distinct roles within the C. merolae spliceosome or function under different cellular and environmental conditions. The importance of these findings stems beyond the spliceosome as they inform on how a small number of residue changes in a protein can have a large impact on protein stability.