Degradation of apolipoprotein II mRNA occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-noncoding region

Degradation intermediates of the estrogen-regulated apolipoprotein (apo) II mRNA were identified by S1 nuclease mapping and primer extension analysis. S1 mapping of poly(A)-RNA detected a series of mRNAs truncated at specific sites in the 3'-noncoding region. Many of these sites were also detected by primer extension analysis indicating that truncated molecules resulted from endonucleolytic cleavage in the 3'-noncoding region. Identical cleavage sites were seen with RNA from estrogen-treated animals or from animals withdrawn from hormone under conditions where apoII mRNA degraded in the slow (t1/2 = 13 h) or rapid (t1/2 = 1.5 h) decay mode. No differences were seen in poly(A) tail length or heterogeneity among these conditions. These results indicate that the estrogen-induced alteration in apoII mRNA turnover does not involve a new pathway of degradation, but, more likely, involves an increased targeting of the mRNA for degradation by a preexisting pathway. These data are consistent with a mechanism in which the initial step in apoII mRNA degradation is an endonucleolytic cleavage in the 3'-noncoding region without prior removal of the poly(A) tail. The endonucleolytic cleavage sites occurred predominantly at 5'-AAU-3' or 5'-UAA-3' trinucleotides found in single-stranded domains in a secondary structure model of the naked mRNA (Hwang, S-P. L., Eisenberg, M., Binder, R., Shelness, G. S., and Williams, D. L. (1989) J. Biol. Chem. 264, 8410-8418). The structure of the 3'-noncoding region in polyribosomal messenger ribonucleoprotein was examined by titrations of liver homogenates with dimethyl sulfate and cobra venom RNase. The results suggest that the typical cleavage site is a 5'-AAU-3' or 5'-UAA-3' trinucleotide in an accessible single-stranded loop domain. Single-stranded domains alone or accessible domains alone are not sufficient for cleavage. Similarly, 5'-AAU-3' or 5'-UAA-3' trinucleotides alone are not sufficient for cleavage. Localization of these trinucleotides to accessible single-stranded domains in the polyribosomal messenger ribonucleoprotein may provide the specificity for cleavage during targeted degradation.     

Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure

UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site.     

A 32-kilodalton protein binds to AU-rich domains in the 3'' untranslated regions of rapidly degraded mRNAs.

An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.     

Defining a novel ribonucleotide reductase r1 mRNA cis element that binds to an unique cytoplasmic trans-acting protein.

Ribonucleotide reductase is a highly regulated rate-limiting enzyme activity in DNA synthesis, responsible for reducing ribonucleotides to their deoxyribonucleotide forms. Using 3'-end labeled RNA and band-shift and UV cross-linking analyses, we have identified a cis-element, 5'-CAAACUUC-3', within the 3'-untranslated region of the mammalian ribonucleotide reductase R1 mRNA, which binds a cytoplasmic protein in BALB/c 3T3 mouse cells, to form a 57 kDa RNA-protein complex. Sequence-specific binding was observed, and binding was prevented by several different mutations within the cis-element. We suggest that this cis-trans interaction plays a role in R1 mRNA stability.     

A host factor that binds near the termini of hepatitis B virus pregenomic RNA.

The terminal regions of hepatitis B virus (HBV) pregenomic RNA (pgRNA) harbors sites governing many essential functions in the viral life cycle, including polyadenylation, translation, RNA encapsidation, and DNA synthesis. We have examined the binding of host proteins to a 170-nucleotide region from the 5' end of HBV pgRNA; a large portion of this region is duplicated at the 3' end of this terminally redundant RNA. By UV cross-linking labeled RNA to HepG2 cell extracts, we have identified a 65-kDa factor (p65) of nuclear origin which can specifically bind to this region. Two discrete binding sites were identified within this region; in vitro cross-competition experiments suggest that the same factor binds to both elements. One binding site (termed UBS) overlaps a portion of the highly conserved stem-loop structure (epsilon), while the other site (termed DBS) maps 35 nucleotides downstream of the hexanucleotide polyadenylation sequence. Both binding sites are highly pyrimidine rich and map to regions previously found to be important in the regulation of viral polyadenylation. However, functional analysis of mutant binding sites in vivo indicates that p65 is not involved in the polyadenylation of HBV pgRNA. Potential roles for the factor in viral replication in vivo are discussed.     

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

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.     

Isolation and characterization of a folate receptor mRNA-binding trans-factor from human placenta. Evidence favoring identity with heterogeneous nuclear ribonucleoprotein E1

The interaction of an 18-base cis-element in the 5'-untranslated region of human folate receptor (FR)-alpha mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539-25547). This trans-factor was isolated to apparent homogeneity as a 43- and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA cis-element in the 3'-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as alphaCP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.     

The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region.

Initiation of translation of poliovirus RNA by ribosomal entry into an internal segment of the 742-nucleotide (nt)-long 5' nontranslated region involves trans-acting factors, including p57, a 57-kDa polypeptide which has been identified as the pyrimidine tract-binding protein (PTB). A UV cross-linking assay was used to compare the RNA-binding properties of the p57 present in various mammalian cytoplasmic extracts with those of purified murine p57 and recombinant human PTB. Three noncontiguous p57-binding sites were located within the poliovirus 5' nontranslated region, between nt 70 and 288, and 443 and 539 (domain V), and 630 and 730. With the same assay, a novel 34-kDa polypeptide was identified that bound nt 1 to 629 specifically. A single A-->G substitution of nt 480 which attenuates poliovirus did not alter UV cross-linking of p57 to domain V. Although UV cross-linking of p57 to the internal ribosome entry site was specifically reduced by competition with poly(U) but not by competition with poly(C), poly(G), and poly(A) homoribopolymers, the presence of a polyuridine tract was not a sufficient determinant for binding of RNA to the p57 present in cytoplasmic extracts, nor was the polypyrimidine tract downstream of domain V necessary for binding to this site.     

Direct evidence for interaction between COOH-terminal regions of cytochrome b558 subunits and cytosolic 47-kDa protein during activation of an O(2-)-generating system in neutrophils.

Cytochrome b558 in phagocytes is a transmembrane protein composed of large and small subunits and considered to play a key role in O2- generation during the respiratory burst. The COOH-terminal regions of the cytochrome subunits protrude to the cytoplasmic side and are assumed to be the sites for association with cytosolic components to form an active O(2-)-generating complex (Imajoh-Ohmi, S., Tokita, K., Ochiai, H., Nakamura, M., and Kanegasaki, S. (1992) J. Biol. Chem. 267, 180-184). We show here that two synthetic peptides corresponding to the COOH-terminal region of each subunit inhibit NADPH-dependent oxygen uptake induced by sodium dodecyl sulfate (SDS) in a cell-free system consisting of plasma membrane and cytosol. The inhibition was observed when either peptide was added to the system before, but not after, the activation with SDS suggesting that interaction between the COOH-terminal regions of the cytochrome subunits and cytosolic components is important for the assembly and the activity of the O(2-)-generating system. Using the cross-linking reagent dimethyl 3,3'-dithiobis-propionimidate, we found that the cytosolic 47-kDa protein, an essential component of the O(2-)-generating system, interacted with the synthetic peptides in the presence of SDS. In addition to the 47-kDa protein, a 17-kDa protein was found to be associated with the peptide corresponding to the COOH-terminal region of the small subunit. These results indicate that the cytosolic COOH-terminal regions of cytochrome b558 subunits are the binding sites for both the cytosolic 47-kDa protein and the 17-kDa protein and that the binding takes place during activation of the system.     

Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species

The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3'-untranslated region of the hamster beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for beta ARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.     

Association of Guide RNA Binding Protein gBP21 with Active RNA Editing Complexes in Trypanosoma brucei

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.