Subcellular structure containing mRNA for β subunit of mitochondrial H+-ATP synthase in rat hepatocytes is translationally active

Abstract

We have recently reported that the nuclear-encoded mRNA for the beta subunit of mitochondrial H+-ATP synthase (beta-mRNA) is localized in rounded, electron-dense clusters in the cytoplasm of rat hepatocytes. Clusters of beta-mRNA are often found in close proximity to mitochondria. These findings suggested a role for these structures in controlling the cytoplasmic expression and sorting of the encoded mitochondrial precursor. Here we have addressed the question of whether the structures containing beta-mRNA are translationally active. For this purpose a combination of high-resolution in situ hybridization and immunocytochemical procedures was used. Three different co-localization criteria showed that beta-mRNA-containing structures always revealed positive immunoreactive signals for mitochondrial H+-ATP synthase (F1-ATPase), ribosomal and hsc70 proteins. Furthermore, clusters show evidence in situ of developmental changes in the translational efficiency of the beta-mRNA. These findings suggest that structures containing beta-mRNA are translationally active irrespective of their cytoplasmic location. The immunocytochemical quantification of the cytoplasmic presentation of hsc70 in the hepatocyte reveals that approx. 86% of the protein has a dispersed distribution pattern. However, the remaining hsc70 is presented in clusters of which only half reveal positive hybridization for beta-mRNA. The interaction of hsc70 with the beta-F1-ATPase precursor protein is documented by the co-localization of F1-ATPase immunoreactive material within cytoplasmic clusters of hsc70 and by the co-immunoprecipitation of hsc70 with the beta-subunit precursor from liver post-mitochondrial supernatants. Taken together, these results suggest a role for hsc70 in the translation/sorting pathway of the mammalian precursor of the beta-F1-ATPase protein.

Control of the Translational Efficiency of β-ATPase mRNA Depends on the Regulation of a Protein That Binds the 3′ Untranslated Region of the mRNA

“…These structures have been shown to contain 60S ribosomal proteins as well as molecular chaperones (54). Likewise, the stability of ␤-F1-ATPase mRNA is controlled throughout liver development (30).…”

“…Furthermore, using a combination of in situ hybridization and immunocytochemical procedures, we have provided evidence that cytoplasmic ␤-mRNA clusters are the subcellular sites involved in the control of the translation of the transcript during development (54). In light of the close proximity between ␤-mRNA clusters and mitochondria, a role for this structure in fast-tracking the sorting of the precursor protein to its mitochondrial destination has been suggested (20,54; for a review see reference 38).…”

Localized translation near the mitochondrial outer membrane: An update

“…This result suggested immediate translocation through import-sites on the mitochondria. 37 A few years later, several seminal studies from the laboratory of C. Jacq demonstrated that many more mRNAs are localized to the vicinity of the mitochondria in the yeast model system. Yeast cells were fractionated by differential centrifugation and the mRNAs that were associated with the mitochondrial fraction were identified.…”

A Conserved Mechanism for Controlling the Translation of β-F1-ATPase mRNA between the Fetal Liver and Cancer Cells

“…The nuclear-encoded ␤-F1-ATPase mRNA has been recently shown to be localized in defined cytoplasmic structures of the hepatocyte that appear to be responsible for controlling the cytoplasmic metabolism of the transcript (31,36), as well as the delivery of the encoded precursor protein to mitochondria (36,37). Most described cis-acting sequences involved in the localization and regulation of mRNA turnover are placed within the 3Ј-UTR of the mRNAs (38 -41).…”