Chapter 3 Purification and Analysis of the Decapping Activator Lsm1p‐7p‐Pat1p Complex from Yeast
Introduction
Turnover of mRNA is an important control point in gene expression, and misregulation of mRNA stability can result in a variety of diseases (Cheadle et al., 2005, Fraser et al., 2005, Hollams et al., 2002, Seko et al., 2006, Steinman, 2007, Tharun and Parker, 2001b). Two major decay pathways conserved in eukaryotes are the 5′ to 3′‐decay pathway and the 3′ to 5′‐decay pathway. Poly(A) shortening is the first step in both pathways, leading to the generation of oligoadenylated mRNA from polyadenylated mRNA. The oligoadenylated mRNA is then degraded in a 3′ to 5′‐exonucleolytic fashion by the exosome (3′ to 5′‐pathway), or is decapped and then degraded by 5′ to 3′‐exonucleolysis (5′ to 3′‐pathway) (Coller and Parker, 2004, Meyer et al., 2004).
Studies determining the characteristics of the components of the cellular mRNA decay machinery are pivotal to the understanding of the mechanism of mRNA decay. Such studies necessitate the use of both genetic and biochemical approaches, because each of these approaches by itself has certain limitations that can be complemented by one another. Biochemical studies on several mRNA decay factors have provided valuable insight in the past on the mechanism of mRNA decay, especially with regard to deadenylation and decapping (Dehlin et al., 2000, Jiao et al., 2006, Khanna and Kiledjian, 2004, Korner and Wahle, 1997, Tucker et al., 2002).
In the 5′ to 3′‐pathway, decapping is a crucial rate determining step that permits the degradation of the body of the message. The highly conserved Lsm1p‐7p‐Pat1p complex is required for normal rates of decapping in vivo (Boeck et al., 1998, Bouveret et al., 2000, Tharun et al., 2000). Earlier in vivo studies suggesting that this complex selectively associates with oligoadenylated mRNPs and facilitates their decapping called for a thorough in vitro biochemical analysis of the purified complex to determine its RNA binding characteristics (Tharun and Parker, 2001a, Tharun et al., 2000). Therefore, we purified this complex from the yeast Saccharomyces cerevisiae and carried out RNA binding studies (Chowdhury et al., 2007).
Section snippets
Purification of the Lsm1p‐7p‐Pat1p Complex
In principle, the purified eukaryotic protein needed for an in vitro biochemical study can be obtained by purification from the original eukaryotic source or from Escherichia coli engineered to express that protein. If no critical posttranslational modifications are required for the protein activity, often the preferred method is to express the protein in E. coli, because it is easy to obtain large quantities of highly purified protein that way. Simple protein complexes composed of a few
Analysis of RNA Binding by the Lsm1p‐7p‐Pat1p Complex
The method we use for determining the RNA binding activity of the Lsm1p‐7p‐Pat1p complex is the gel mobility shift assay which is a sensitive and widely used method for studying nucleic acid–protein interactions (Black et al., 1998). Here the labeled nucleic acid is incubated with the nucleic acid binding protein, and the binding reaction is allowed to reach the equilibrium. The protein‐bound nucleic acid molecules in the reaction mixture are then resolved from the unbound nucleic acid
Concluding Remarks
Purification of the yeast Lsm1p‐7p‐Pat1p complex and analysis of its RNA binding properties with the procedures described previously revealed that it has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter (and unadenylated RNA) (Chowdhury et al., 2007). This defines an important RNA binding characteristic of this complex and is consistent with earlier studies, suggesting that it
Acknowledgments
This work was supported by NIH grant (GM072718) and USUHS intramural grant (C071HJ).
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