Assembly of the ribonucleoprotein complex containing the mRNA of the beta-subunit of the mitochondrial H+-ATP synthase requires the participation of two distal cis-acting elements and a complex set of cellular trans-acting proteins.
The mRNA encoding the beta-subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) is localized in an approx. 150 nm structure of the hepatocyte of mammals. In the present study, we have investigated the cis- and trans-acting factors involved in the generation of the ribonucleoprotein complex containing beta-F1-ATPase mRNA. Two cis-acting elements (beta1.2 and 3'beta) have been identified. The beta1.2 element is placed in the open reading frame, downstream of the region encoding the mitochondrial pre-sequence of the protein. The 3'beta element is the 3' non-translated region of the mRNA. Complex sets of proteins from the soluble and non-soluble fractions of the liver interact with the beta1.2 and 3'beta elements. A soluble p88, present also in reticulocyte lysate, displays binding specificity for both the cis-acting elements. Sedimentation and high-resolution in situ hybridization experiments showed that the structure containing the rat liver beta-F1-ATPase mRNA is found in fractions of high sucrose concentration, where large polysomes sediment. Treatment of liver extracts with EDTA promoted the mobilization of beta-F1-ATPase mRNA to fractions of lower sucrose concentration, suggesting that the structure containing beta-F1-ATPase mRNA is a large polysome. Finally, in vitro reconstitution experiments with reticulocyte lysate, using either the full-length, mutant or chimaeric versions of beta-F1-ATPase mRNA, reveal that the assembly of the beta-F1-ATPase mRNA polysome requires the co-operation of both the cis-acting mRNA determinants. The present study illustrates the existence of an intramolecular RNA cross-talking required for the association of the mRNA with the translational machinery.
Ricart
Javier
J
Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Izquierdo
José M
JM
Di Liegro
Carlo M
CM
Cuezva
José M
JM
D020413:3' Untranslated Regions; D000818:Animals; D015153:Blotting, Western; D005260:Female; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D011247:Pregnancy; D006180:Proton-Translocating ATPases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D006180:Proton-Translocating ATPases