Cross‐linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells

Ruždijić, Sabera; Bag, Jnanankur; Sells, Bruce H.

doi:10.1111/j.1432-1033.1984.tb08277.x

Cited by 22 publications

(13 citation statements)

References 32 publications

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“…These mRNAs were found on small polysomes or mRNPs and their translation rates were stimulated by CHX. The suggestion was made, based on work in many laboratories (11,33,36,37,42), that the low translational efficiency of these mRNAs is due to an interaction with a repressor protein(s) which hinders the accessibility of mRNA to an mRNA-discriminatory factor. According to this model, CHX treatment actually stimulates the synthesis of proteins encoded by these mRNAs by making them more effective competitors for the limiting component of initiation.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

Rao¹,

Slobin²

1987

Mol. Cell. Biol.

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When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.

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Section: Discussionmentioning

confidence: 99%

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

Rao¹,

Slobin²

1987

Mol. Cell. Biol.

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“…Thus the polysomal mRNP proteins are bound to the globin mRNA during the isolation procedure and retain their binding specificity for mRNA in the assay used here. Proteins of comparable electrophoretic mobility were detected in polysomal mRNP by ultraviolet crosslinking experiments [7,8,10,111, suggesting that the proteins detected here are those which interact directly with the mRNA strand also in the mRNP. Proteins in the M,-45000 range found here were dctected up to now in several cell types by ultraviolet crosslinking [4,8,9] but not in polysomal mRNP from rabbit reticulocytes [8].…”

Section: Discussionmentioning

confidence: 53%

“…The protein component of these messenger ribonucleoproteins (mRNP) may contribute to the translational activity and the stability of a given mRNA 11-31, The direct interaction of the mRNP proteins with the mRNA was shown unambiguously by ultraviolet-light-induced photochemical crosslinking in vivo and in vitro 110, 111 and by affinity chromatography [12]. These techniques revealed a set of proteins binding directly to translated mRNA with apparent M , ranging over 15000-135000 of which a group ranging over M , 52000-94000 is found in all cells investigated so far [I, [4][5][6][7][8][9][10][11][12]. These proteins, together with others which are bound more transiently [13 -151, and the mRNA make up the polysomal mRNP.…”

mentioning

confidence: 99%

Binding of globin mRNA, β‐globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein

Görlach

Hermann

Schwemmle

et al. 1989

European Journal of Biochemistry

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The binding of rabbit globin mRNA, in-vitro-generated P-globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of P-globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)-free j-globin mRNA. A protein of M , 90 000 binds specifically the 3'-nontranslated trailer of the poly(A)-free P-globin mRNA and not the poly(A)-containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of M , 72000, which is most likely the rabbit poly(A)-binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes usedThe mRNAs of eucaryotic cells are complexed with specific proteins [1]. The protein component of these messenger ribonucleoproteins (mRNP) may contribute to the translational activity and the stability of a given mRNA 11-31, The direct interaction of the mRNP proteins with the mRNA was shown unambiguously by ultraviolet-light-induced photochemical crosslinking in vivo [4-91 and in vitro 110, 111 and by affinity chromatography [12]. These techniques revealed a set of proteins binding directly to translated mRNA with apparent M , ranging over 15000-135000 of which a group ranging over M , 52000-94000 is found in all cells investigated so far [I, 4-12]. These proteins, together with others which are bound more transiently [13 -151, and the mRNA make up the polysomal mRNP. The best characterized polysomal mRNP proteins so far are the poly(A)-binding protein [16-211 and the cap-binding protein of M , 24000 [22 -311. Other proteins interacting directly with the mRNA were shown to bind internally between the cap and the poly(A) tail of translated mRNAs [11, 311. However, reports on specific protein-binding sites as determined by nuclease resistant sites of niRNAs are contradictory [32 -361. More recently, a method originally developed for the study of DNA/protein interactions [37] was successfully used to identify and characterize viral RNA-binding proteins [38], a protein binding to the 3' splice site of mammalian pre-mRNA introns [39, 401 and certain hnRNP proteins [41]. This method involves separation of the proteins in question by SDSjPAGE and transfer of the separated proteins onto nitrocellulose filters prior to the binding of labelled nucleic acids to the proteins.We have investigated polysomal mRNP proteins from rabbit reticulocytes by this technique using rabbit globin mRN A, segments of P-globin mRNA transcribed in vitro and RNA homopolymers as RNA probes, in order to characterize the RNA-binding properties of the polysomal mRNP proteins and to look for s...

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“…Recently, Ruzdijic et al (27) have analyzed proteins cross-linked to histone mRNA of HeLa cells by using a different hybrid selection procedure, and they have arrived at a similar conclusion.…”

Section: Discussionmentioning

confidence: 85%

Hybrid selection of messenger ribonucleoprotein for serum albumin: analysis of specific message-bound proteins.

Johnson¹,

Ilan²

1985

Proc. Natl. Acad. Sci. U.S.A.

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The messenger RNA for chicken serum albumin together with its specific binding proteins was purified by hybrid selection using filter-bound cloned albumin cDNA. Under conditions where hybridization of the protein-mRNA complex was specific for the immobilized cDNA sequence, no dissociation of the complex into its protein and RNA components was apparent. Electrophoretic analysis of albumin mRNA-specific binding proteins showed three major bands.Two of these were prominent in total poly(A) messenger ribonucleoprotein. The third band was of much greater relative intensity in the albumin mRNA-specific proteins than in total poly(A) messenger ribonudeoprotein. The results suggest that the proteins bound to albumin mRNA represent only a subset of the total population of poly(A) mRNA-associated protein.In eukaryotic cells, mRNA is found complexed with a variety of proteins and is more appropriately termed messenger ribonucleoprotein (mRNP) (1). Many investigators have shown that mRNPs are found both associated with polysomes (i.e., are being actively translated) and in the postpolysomal ("free") fraction (2). The relationship between free and polysome-associated mRNPs is not well understood and may differ from system to system. The protein components of mRNPs from many cell types have been analyzed, and in most cases differences are found between the free and polysome-associated fractions (3). Although no functions for proteins from mRNPs are known with certainty, with the exception of the poly(A) organizing protein (4), strong circumstantial evidence suggests that at least some of these proteins are accessory factors for protein synthesis or are involved in other aspects of translational regulation (5, 6).One outstanding difficulty in the study of mRNPs has been the inability, except in highly specialized systems, to analyze the proteins bound to a particular mRNA sequence. Thus, it is not known if the same mRNA sequence binds the same set of proteins in both the free and the polysome-associated fractions or, conversely, if different mRNA sequences in the same fraction bind the same set of proteins. This problem became important in our laboratory since we observed that the protein components of mRNPs ofimmature cockerel liver changed markedly in response to estradiol stimulation (7). Estradiol stimulation also profoundly alters the mRNA sequence population (8), rates of protein synthesis for specific proteins (9), and other aspects of hepatic metabolism. In this report, we show that one abundant chicken liver mRNP, that for serum albumin, may be purified by hybridization to cloned albumin cDNA sequences and that the proteins bound to it appear to be a limited subset ofthe population ofproteins associated with total mRNPs. MATERIALS AND METHODSLiver poly(A) mRNA was isolated from estradiol-injected cockerels according to Chirgwin et al. (10). Poly(A) mRNPs were isolated as described (7). cDNAs for chicken serum albumin and very low density lipoprotein II apoprotein (apoVLDL II) (a low molecular weight estradiol-...

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Cross‐linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells

Cited by 22 publications

References 32 publications

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

Binding of globin mRNA, β‐globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein

Hybrid selection of messenger ribonucleoprotein for serum albumin: analysis of specific message-bound proteins.

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