An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro.
The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-Globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ribonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, alphaCP1 and alphaCP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have devised an in vitro mRNA decay assay which reproduces the alpha-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the alpha-complex by sequestration of alphaCP1 and alphaCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the alpha-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between alphaCP1 and alphaCP2 with PABP suggests that the alpha-complex can directly interact with PABP. Therefore, the alpha-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.
Wang
Z
Z
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA.
Day
N
N
Trifillis
P
P
Kiledjian
M
M
D020413:3' Untranslated Regions; D000818:Animals; D002417:Cattle; D004268:DNA-Binding Proteins; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D020014:K562 Cells; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C094604:PCBP1 protein, human; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D005914:Globins