Chapter 3 Purification and Analysis of the Decapping Activator Lsm1p‐7p‐Pat1p Complex from Yeast

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Abstract

Biochemical analysis of the components of the mRNA decay machinery is crucial to understand the mechanisms of mRNA decay. The Lsm1p‐7p‐Pat1p complex is a key activator of decapping in the 5′ to 3′‐mRNA decay pathway that is highly conserved in all eukaryotes. The first step in this pathway is poly(A) shortening that is followed by the selective decapping and subsequent 5′ to 3′‐exonucleolytic degradation of the oligoadenylated mRNAs. Earlier studies suggested that the Lsm1p‐7p‐Pat1p complex preferentially associates with oligoadenylated mRNAs and facilitates their decapping in vivo (Tharun and Parker, 2001a; Tharun et al., 2000). They also showed that the Lsm1p through Lsm7p and Pat1p are involved in protecting the 3′‐ends of mRNAs in vivo from trimming (He and Parker, 2001). Therefore, to gain better insight into the biologic function of the Lsm1p‐7p‐Pat1p complex, it is important to determine its in vitro RNA binding properties. Here I describe the methods we use in my laboratory for the purification and in vitro RNA binding analysis of this complex from the budding yeast Saccharomyces cerevisiae. Purification was achieved with tandem affinity chromatography using a split‐tag strategy. This involved use of a strain expressing FLAG‐tagged Lsm1p and 6×His‐tagged Lsm5p and purification by a two‐step procedure with an anti‐FLAG antibody matrix followed by a Ni–NTA matrix. The purified complex was analyzed for its RNA binding properties with gel mobility shift assays. Such analyses showed that this complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs and that it binds near the 3′‐ends of RNAs (Chowdhury et al., 2007). These observations, therefore, highlighted the importance of the intrinsic RNA binding properties of this complex as key determinants of its in vivo functions.

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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