Possible involvement of messenger RNA-associated proteins in protein synthesis.

Abstract

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization . The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system . Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP ; deproteinized mRNA however was not translated . Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated . Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP .Many investigations support the concept that eukaryotic messenger RNA is associated with various proteins as messenger ribonucleoprotein particles (mRNP) (1-8) . Two classes of mRNP can be isolated from the cytoplasm, one that consists of free mRNP not bound to ribosomes, and the other that is present in polyribosomes (1,3,6,9,10) . The physiological importance of stable protein-mRNA interactions and even the existence of mRNP have sometimes been questioned, because mRNP and deproteinized mRNA stimulate the cell-free translation system equally well (11-15) . Furthermore, earlier studies by 17) indicated that the cytosol contains soluble proteins with affinity for RNA, raising the possibility that mRNPs are artifacts of cell fractionation (see discussion in Kumar and Pederson (18]). On the other hand, mRNPs can be shown to remain intact under conditions of ionic strength where nonspecific RNA-protein complexes dissociate (14,(19)(20)(21)(22)(23) . Moreover, RNA-protein cross-linking experiments indicate that mRNA is complexed with proteins in situ (24-26) .These conflicting observations can be reconciled by the hypothesis that RNA-protein associations do occur in vivo, but that the mRNP-proteins are also present as soluble RNAbinding proteins . This view is compatible with the metabolic behavior of mRNP, which indicates that the proteins shuttle on and off the mRNA (27)(28)(29) . In addition, recent data from this laboratory (30-32), as well as from others (33,34), have shown that in fact some of the RNA-binding proteins have the same electrophoretic mobilities as the proteins isolated in association with mRNAs as native mRNP in the same cells .Clearly, the possible role of mRNP proteins in translation cannot be investigated if one uses a translation system in which THE JOURNAL OF CELL BIOLOGY " VOLUME 93 JUNE 1982 893-898 c0 The Rockefeller University Press " 0021-9525/82/06/0893/06$1 .00 an extensive supply of these proteins is present in soluble form . Therefore, we have devised a method for eliminating these proteins from an ascites cell lysate, by passing the lysate through a RNA affinity column (31,32) . This treatment makes translation in the lysate dependent on protein factors that bind to the column, or on some ...

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

“…If this is the case, then the mRNA-associated proteins themselves should be able to serve as translation factors; indeed, Schmid et al reported that isolated mRNP stimulated translation in a factor-dependent cell-free trans-* Corresponding author. lation system, whereas deproteinized mRNA did not (34). This finding is the most direct evidence to date for a translational role for the proteins, and it would be conclusive except for uncertainty as to the purity of the isolated mRNP.…”

Prosomes discriminate between mRNA of adenovirus‐infected and uninfected HeLa cells

“…Bound poly(A) ÷ mRNA was eluted with low salt buffer, precipitated with ethanol and stored at -20°C for further analysis [14].…”

Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

“…Although mRNP proteins are hypothesized to perform roles in translational regulation, the function of most of them remain unknown (18,31,32,37,43,(54)(55)(56). To date, the four cap-binding proteins (eIF-4E and the three proteins of the eIF-4F complex) and the poly(A)-binding proteins (PABPs) are the only mRNP proteins that have been identified across species boundaries.…”