Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles

Abstract

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphytococcus auLeuz nuclease (EC 3. 1. 4. 7.). 25 % of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine.Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in wvtAo iodine-labelled 9 S globin mRNA and RNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 $ mRNP. INTRODUCTIONMessenger RNA in the reticulocyte cytoplasm is associated with proteins to form messenger ribonucleoprotein (mRNP) complexes. In the case of globin m RNA 2 kinds of mRNP complexes have been previously described: the actively

Ribonucleotide sequences non-adjacent to poly(A) participate in the poly(A)-protein complex in 15S duck globin mRNP particles

“…We have recently shown that in 15 S duck globin mRNP, 25 % of the mRNA sequences are protected by proteins against staphylococcal nuclease digestion, and that proteins interact non-randomly with the mRNA sequences, protecting specific mRNA fragments (22). Similar results were obtained for nuclear pre-mRNP particles (23).…”

“…Fingerprint analysis : Electrophoresis in first dimension was on cellulose acetate strips at 4,000 volts for 60 minutes using a buffer containing 5 % acetic acid, 2 mM EDTA, 7 M urea adjusted to pH 3.5 with pyridine. Second dimension analysis was homochromatography on DEAE-cellulose thin layer plates using homomixture C, 650 C, 6 hours, as described previously (22), according to Brownlee's procedure (25). Affinity chromatography : mRNA was chromatographed on oligo-(dT)-cellulose as described by others (26)(27), with the difference that binding of poly(A+)…”

“…In vitro labelin of RNA with 32 at the 5'-end was carried out with y-(32P)ATP in the reaction catalysed by the polynucleotide kinase (30). Enzymatic digestions and phenol extraction were performed as described previously (22), and autoradiographs were performed at -70°C.…”

The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific ScRNA and a characteristic set of proteins.

“…Labelling was carried out with 32P-labelled pCp in a reaction catalyzed by T4 RNA ligase according to Peattie (1979). Enzymatic digestion of ScRNA with RNAse Tl was performed as described earlier (Goldenberg et al, 1979). Electron microscopy A drop of the appropriate sucrose gradient fractions (cf., Figure 1) was put onto freshly glow-discharged and carbon-coated copper grids.…”

The gene and the genon concept: a functional and information‐theoretic analysis

“…All types of RNA in the cell are covered by proteins (1:3 ratio in mRNPs). In the case of mRNA, it was shown by electron microscopy (Dubochet et al , 1973) that proteins are aligned along the entire length of the RNA molecules (and it is thus likely also for pre‐mRNA), protecting specific oligomotifs from degradation by RNase (Goldenberg et al , 1979). Only in rare cases the oligonucleotide sequence that binds a given regulatory protein is known; in addition to the poly(A)‐binding protein (PABP), one might mention, for example, the IRE‐BP (iron response element binding protein), a protein that binds an oligomotif in the 5′‐side or 3′‐side UTR of the mRNAs for ferritin and transferrin, respectively (Thomson et al , 1999).…”