Identification of a C-terminal Poly(A)-binding Protein (PABP)-PABP Interaction Domain: ROLE IN COOPERATIVE BINDING TO POLY(A) AND EFFICIENT CAP DISTAL TRANSLATIONAL REPRESSION

Eduardo O Melo,Rafael Dhalia,Cezar Martins De Sa,Nancy Standart,Osvaldo P De Melo Neto

doi:10.1074/jbc.m307624200

Abstract

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

Highlights

The poly(A)-binding protein (PABP), bound to the 3؅ poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability
In vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5؅-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides
The poly(A)-binding protein (PABP)1 bound to the 3Ј poly(A) tail of eukaryotic mRNAs is recognized as an important if not an essential component of the apparatus required for mRNA translation

Summary

EXPERIMENTAL PROCEDURES

Cyclin Reporter Plasmids—The cyclin mRNAs described in this study were based on the Xenopus cyclin A cDNA in the plasmid pTZ18R, which contained a tract of 61 A residues, located 41 nucleotides from the 5Ј end of cyclin mRNA [25]. In Vitro Transcription and Translation—Transcriptions were performed as described previously [25], using T7 RNA polymerase with SalI (cyclin) or BamHI (PABP) linearized plasmid DNAs. For the translation reactions, the rabbit reticulocyte cell-free system (Promega) was treated with micrococcal nuclease to render it mRNA-dependent. Labeled human eIF4G was obtained by subcloning the XbaI/HindIII fragment from plasmid pSK-HFC1 [41] into the same sites of the pBluescript KS vector (Stratagene), followed by linearization with HindIII and in vitro transcription and translation as described above. MS2 Tethering Constructs—The reporter plasmids MSC-15 and MSA-15 were obtained by digestion of the MSC-GH and MSA-GH plasmids [43] with XbaI/HindIII and ligation of the larger fragment to the BamHI/HindIII fragment encoding the CAT gene from plasmid pSV2-CAT Both the vector and insert DNAs were in-filled with Klenow DNA polymerase prior to ligation. Probes were labeled with [␣-32P]dCTP using the Megaprime kit (Amersham Biosciences)

RESULTS

DISCUSSION

Melo Neto