Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7-Pat1 complex.
Decapping is a critical step in the conserved 5'-to-3' mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7-Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3'-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3'-end protection but unaffected in Lsm1-7-Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3'-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7-Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3'-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.
Chowdhury
Ashis
A
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Tharun
Sundaresan
S
D000483:Alleles; D047468:Immunoprecipitation; D009154:Mutation; D011485:Protein Binding; D039381:RNA Cap-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012315:RNA Caps; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C114069:LSM1 protein, S cerevisiae; C103266:PAT1 protein, S cerevisiae; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins