Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.

Abstract

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.It is becoming increasingly clear that the proteins associated with cytoplasmic mRNA in eucaryotic cells associate in the cytoplasm with already existing mRNA rather than coming out of the nucleus along with newly synthesized mRNA and that they function in translation or in some related cytoplasmic process such as determining mRNA stability. This laboratory and others showed that these proteins differ dramatically from those of nuclear RNA-protein complexes containing mRNA precursors (21,24,35). Even cytoplasmic polyadenylate [poly(A)]-protein complexes differ in structure and composition from their nuclear counterparts (1,35,39). The labeling kinetics of the cytoplasmic mRNA-associated proteins were consistent with cytoplasmic addition to mRNA (13). These findings make it unlikely that they are packaging or transport proteins.The mRNA-associated proteins are similar in size, charge, and amino acid composition to soluble RNA-binding proteins which can serve as initiation and elongation factors in translation (26,28,36,37,40). Furthermore, we showed that the proteins of polyribosomal mRNA-protein complexes (mRNP) continue to be synthesized and to become associated with mRNA in the absence of mRNA synthesis (13 lation system,...

The poly(A)‐binding protein facilitates in vitro translation of poly(A)‐rich mRNA

“…Comparison of the translational efficiency in vitro between phenol-extracted globin mRNA and the corresponding mRNP has shown no difference [23]. In view of the evolutionary conservation of the poly(A)-binding protein and its abundance in a free form in the cytoplasm [20, 241, the possibility that exogenous mRNA immediately reconstitutes an mRNP when added to the cell-free lysate had to be taken into consideration [25].We have investigated the possible role of the poly(A)-binding protein in protein synthesis by analysing the effect of homo-polyribonucleotides on translation, when added to the lysate in presence of polyadenylated and non-adenylated mRNAs. We reasoned that, if the interaction between the poly(A) region of mRNA and its binding protein is important, addition of exogenous poly(A) may have an effect by limiting the availability of unbound protein.…”

“…Comparison of the translational efficiency in vitro between phenol-extracted globin mRNA and the corresponding mRNP has shown no difference [23]. In view of the evolutionary conservation of the poly(A)-binding protein and its abundance in a free form in the cytoplasm [20, 241, the possibility that exogenous mRNA immediately reconstitutes an mRNP when added to the cell-free lysate had to be taken into consideration [25].…”

Eukaryotic elongation factor Tu is present in mRNA‐protein complexes

“…Preparation of rabbit reticulocyte lysate, UV crosslinking, and [14C]-formaldehyde labeling was done as described [10]. eEF-Tu was purified as in [11].…”

“…The spots corresponding to these species were readily detected in the 2D gels ( fig.lA). Some bands showed multiple spots or streaking in the first dimension suggestive of charge isomerism, particularly the 52 and 62 kDa bands, mRNP proteins also contain species of 68 and 65 kDa which are not always resolved in 1D SDS-PAGE [10]. However, these species gave 2 well-separated spots in a 2D gel ( fig.lA).…”

Characterization of mouse reticulocyte free globin mRNP

“…Gradient fractions containing cross-linked either polysomal or free mRNP were pooled and purified as described in [8,13,14]. The yield of purified total free mRNP (region I and II) was estimated to be -5 ug per 100 mice.…”