The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex.
Repression of poly(A)-binding protein (PABP) mRNA translation involves the binding of PABP to the adenine-rich autoregulatory sequence (ARS) in the 5'-untranslated region of its own mRNA. In this report, we show that the ARS forms a complex in vitro with PABP, and two additional polypeptides of 63 and 105 kDa. The 63 and 105 kDa polypeptides were identified, as IMP1, an ortholog of chicken zip-code binding polypeptide, and UNR, a PABP binding polypeptide, respectively, by mass spectrometry of the ARS RNA affinity purified samples. Using a modified ribonucleoprotein (RNP) immunoprecipitation procedure we further show that indeed, both IMP1 and UNR bind to the ARS containing reporter RNA in vivo. Although both IMP1 and UNR could bind independently to the ARS RNA in vitro, their RNA-binding ability was stimulated by PABP. Mutational analyses of the ARS show that the presence of four of the six oligo(A) regions of the ARS was sufficient to repress translation and the length of the conserved pyrimidine spacers between the oligo(A) sequences was important for ARS function. The ability of mutant ARS RNAs to form the PABP, IMP1 and UNR containing RNP complex correlates well with the translational repressor activity of the ARS. There is also a direct relationship between the length of the poly(A) RNAs and their ability to form a trimeric complex with PABP, and to repress mRNA translation. UV crosslinking studies suggest that the ARS is less efficient than a poly(A) RNA of similar length, to bind to PABP. We show here that the ARS cannot efficiently form a trimeric complex with PABP; therefore, additional interactions with IMP1 and UNR to form a heteromeric RNP complex may be required for maximal repression of PABP mRNA translation under physiological conditions.
Patel
Gopal P
GP
Department of Molecular and Cellular Biology, University of Guelph Guelph, Ontario, Canada, N1G 2W1.
Ma
Shuhua
S
Bag
Jnanankur
J
D020121:5' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006706:Homeostasis; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D039102:Poly(A)-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins / Q000378:metabolism*
D020121:5' Untranslated Regions; C079950:CSDE1 protein, human; D004268:DNA-Binding Proteins; C406144:IGF2BP1 protein, human; D010455:Peptides; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins