A 3' exonuclease that specifically interacts with the 3' end of histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.     

Dinucleotide cap analogue affinity resins for purification of proteins that specifically recognize the 5' end of mRNA

Here we present first dinucleotide affinity resins for purification of proteins that specifically recognize the 5' end of mRNA. Constructed resins possess either a naturally occurring mono- or trimethylated cap or their analogues resistant towards enzymatic degradation, bearing a CH(2) bridge between β and γ position of the 5',5'-triphosphate chain. All cap analogues were attached to a polymer support (EAH-Sepharose) through the carboxylic group that had been generated by derivatization of the 2',3'-cis diol of the second nucleotide in the cap structure with levulinic acid.     

Human mRNA export machinery recruited to the 5' end of mRNA

Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.     

A protein kinase from neutrophils that specifically recognizes Ser-3 in cofilin

Cofilin promotes the depolymerization of actin filaments, which is required for a variety of cellular responses such as the formation of lamellipodia and chemotaxis. Phosphorylation of cofilin on serine residue 3 is known to block these activities. We now report that neutrophils contain a protein kinase that selectively catalyzes the phosphorylation of cofilin on serine 3 (>/=70%) and a nonspecific kinase that recognizes multiple sites in this protein. The selective serine 3 cofilin kinase binds to a deoxyribonuclease I affinity column, whereas the nonspecific cofilin kinase does not. Deoxyribonuclease I forms a very tight complex with actin, and deoxyribonuclease affinity columns have been utilized to identify a variety of proteins that interact with the cytoskeleton. The serine 3 cofilin kinase did not react with antibodies to LIM kinase 1 or 2, which can catalyze the phosphorylation of cofilin in other cell types. The activity of the serine 3 cofilin kinase was insensitive to a variety of selective antagonists of protein kinases but was blocked by staurosporine. This pattern of inhibition is similar to that observed for the kinase that is active with cofilin in intact neutrophils. Thus, neutrophils contain a protein kinase distinct from LIM kinase-1/2 that selectively recognizes serine 3 in cofilin.     

A 60-kDa phosphorylated protein from fetal human bone

A phosphorylated protein was isolated and purified from fetal human bone. Fetal and adult human bones were decalcified with EDTA, and the extract from the fetal bone was fractionated using Q-Sepharose anion exchange chromatography. The fraction containing Ser(P) was purified by Sephacryl S-200 molecular sieving and C4 reverse-phase HPLC. The purified protein had a molecular weight of 60000 on SDS-PAGE, where the protein was stained with Rhodamine-B. The amino acid composition of this protein was different from any other reported phosphorylated proteins in human bone. However, this phosphorylated protein was difficult to detect in the adult bone extract on SDS-PAGE.     

Complete nucleotide sequence of the 5' noncoding region of human alpha-and beta-globin mRNA

The 5' noncoding regions of human alpha-and beta-globin mRNAs, 37 and 50 nucleotides in length, have been sequenced. A variation of the "plus and minus" gel technique described by Brownlee and Cartwright (1977) was used, and the results were cross-checked by the Maxam and Gilbert (1977) procedure. These studies completed the knowledge of all the noncoding region sequences of both mRNAs, and it was then possible to calculate their exact size. Human alpha-and beta-globin mRNAs are 575 and 626 nucleotides in length, excluding the poly(A). Furthermore, because the coding and 3' noncoding regions of the latter were known from previous studies (Marotta et al., 1977; Proudfoot, 1977), the primary structure of human beta-globin mRNA is now complete except for six ambiguities in the coding region. The human and rabbit 5' noncoding region sequences are about 80% homologous. This suggests that they are under a moderate selective pressure.     

Characterization of protein synthesis factors from rabbit reticulocytes

As part of our efforts to characterize eukaryotic translation factors, we have sequenced a number of them chemically and inferred sequences from cDNA clones. To our surprise, there appears to be extensive identity of amino acid sequence in most factors characterized to date in that within mammalian species, usually greater than 99% identity is observed. Extreme examples are rabbit EF-1 alpha which is 100% identical to human EF-1 alpha and rabbit eIF-4AI and eIF-4AII which are 100% identical to mouse eIF-4AI and eIF-4AII for those amino acids sequenced (398/406 and 156/407, respectively). An extended analysis has been made of EF-1 alpha which in rabbit has three different post-translational modifications, dimethyllysine, trimethyllysine and glycerylphosphorylethanolamine. A comparison of the primary structure of EF-1 alpha to E. coli EF-Tu indicates an overall sequence identity of 33%. However, within the amino terminal 180 amino acids (the GTP-binding domain), there are found regions of much greater identity (50/85 = 59%).     

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.     

Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.     

Characterization of a native mRNA containing preinitiation complex from rabbit reticulocytes: RNA and protein constituents

From a rabbit reticulocyte postpolysomal supernatant a fraction has been isolated which is enriched in ribosomal particles sedimenting at 50S. This fraction is efficiently in vitro translated predominantly into α-globin. Besides the RNAs and proteins of the small ribosomal subunit the 50S particle contains α-globin mRNA and additional high molecular weight proteins, most of which correspond to polypeptides of the initiation factors eIF-2 and eIF-3. The 50S particle may represent a native [mRNA·40S·eIF′s·Met-tRNA_f·GTP] complex which may occur in vivo as a translatable intermediate in the initiation sequence.     

A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein

Using a protein blotting method for the detection of nucleic acid binding proteins, we have identified in HeLa cell nuclear extracts an intron binding protein (IBP) that selectively recognizes the 3' splice site region of mammalian pre-mRNAs. The binding site was accurately delineated using oligonucleotides complementary to human beta-globin pre-mRNA. It spans the 3' splice site AG dinucleotide and the crucial polypyrimidine stretch upstream, but includes neither the branchpoint nor the lariat structure. Although the technique used here shows that the binding specificity is an intrinsic property of IBP and does not depend on snRNA-pre-mRNA interactions, it comigrates with U5 snRNP and is immunoprecipitated by anti-Sm antibody. This strongly suggests that IBP belongs to U5 snRNP. We propose that it is involved in one of the earliest steps of the splicing reaction by mediating the interaction of U5 snRNP with the 3' splice site.     

Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein

Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.