Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast.
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 6xHis-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.
Tharun
Sundaresan
S
Department of Biochemistry, Uniformed Services University of Health Sciences, Bethesda, Maryland, USA.
D000818:Animals; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; C114069:LSM1 protein, S cerevisiae; C103266:PAT1 protein, S cerevisiae; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins