Abstract
Pre‐messenger RNA splicing is carried out by a complex and dynamic molecular machine called the spliceosome. Assembly of the spliceosome is a dynamic, complex process in which there are several RNA‐RNA and RNA‐protein rearrangements. As part of this process, Dib1 is an essential protein involved in the assembly of the spliceosome on a pre‐messenger RNA. In yeast, Dib1 is a small, 17 kDa protein that has a thioredoxin‐like fold. Previous studies have found that Dim1, the human homolog of Dib1, possesses protease activity and has the ability to cleave its own C‐terminal tail region. However, how this proteolytic cleavage works as well as the role of this proteolytic activity in splicing is unknown. The purpose of this project is to biochemically characterize the in vitro auto‐cleavage mechanism in order to further understand the unidentified mechanism. Additionally, we aim to determine if truncations of the tail region of Dib1 impact pre‐messenger RNA splicing. The effects of the truncations will be assessed in Saccharomyces cerevisiae. Plasmids were constructed to express Dib1 mutants in yeast to conduct growth assays as well as in E. coli to purify mutant proteins. The effects of mutations on protein stability will be analyzed by circular dichroism and then assessed in auto‐cleavage studies. Our preliminary studies have found that truncations of the Dib1 tail have the most significant effect when the entirety of the tail that is removed in auto‐cleavage is deleted.Support or Funding InformationSan Antonio Area Foundation NIH R15GM120720This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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