An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro

Mol Cell Biol. 1999 Jul;19(7):4552-60. doi: 10.1128/MCB.19.7.4552.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Cattle
  • DNA-Binding Proteins*
  • Globins / genetics*
  • HeLa Cells
  • Heterogeneous-Nuclear Ribonucleoproteins*
  • Humans
  • K562 Cells
  • Poly(A)-Binding Proteins
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Transcription Factors*

Substances

  • 3' Untranslated Regions
  • DNA-Binding Proteins
  • Heterogeneous-Nuclear Ribonucleoproteins
  • PCBP1 protein, human
  • Poly(A)-Binding Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • Transcription Factors
  • Globins