Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA

Abstract

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.

Intrahepatic Induction of Alpha/Beta Interferon Eliminates Viral RNA-Containing Capsids in Hepatitis B Virus Transgenic Mice

“…To further confirm that the HBV and GAPDH transcripts were indeed associated with ribosomes, we treated polyribosomal extracts with 15 mM EDTA, a procedure known to release transcripts from ribosomes (21). As shown in Fig.…”

Translational control in plant stress: the formation of messenger ribonucleoprotein particles (mRNPs) in response to desiccation of Tortula ruralis gametophytes

“…We are attempting to investigate this possibility but it has proven dif®cult to isolate suf®cient quantities of these particles for a detailed reproducible protein analysis because of the large quantities of gametophytic tissue required. Polypeptides associated with polysomal and free mRNP complexes have been described in a number of systems (Bag, 1988;Bag and Sells, 1981;Dreyfuss et al, 1988;Iskakov and Madin, 1994;MacDonald and Williams, 1992). Using in vivo crosslinked preparations of chicken muscle cells, Bag (1984) has demonstrated that the major polypeptides associated with both polyosmal and free mRNPs have Mr that range from 150 000 to 28 000.…”

RNase H/oligonucleotide-directed mRNA purification (ROMP) of apoll mRNA

“…Apoll mRNA can be purified in the polyAfraction after RNase H hydrolysis. Oligonucleotide hybridization and RNase H hydrolysis was performed on total liver RNA, and the results were assessed by Northern blot analysis (5). Panel A is Northern analysis of apoll mRNA.…”

“…The uniformly labelled probe for apoll was made as described (1). The control cDNA probe was 32P-labelled oligo(dT)primed cDNA from non-estrogen treated rooster liver RNA, and thus did not contain apoIl sequences (5). PolyA+ RNA was incubated in the absence of RNase H (lanes a and b), or in the presence of 0.3 units (lanes c-e), 1.0 unit (lanes f-h), or 3.0 units (lanes i-k) of RNase H. Lanes c, f and i represent total RNA after RNase H incubation (T); lanes a, d, g and j are RNA from the flow-through fraction (F), and lanes b, e, h and k from the bound fraction (B) after oligo(dT) cellulose chromatography.…”