The stem-loop structure at the 3'' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability.

Chimeric genes were made by fusing mouse histone genes with a human alpha-globin gene. The genes were introduced into mouse L cells and the stability of the chimeric mRNAs was measured when DNA synthesis was inhibited. An mRNA containing all the globin coding sequences and the last 30 nucleotides of the histone mRNA was degraded at the same rate as histone mRNA.     

Apolipoprotein B mRNA editing: the sequence to the event

Editing of the mRNA coding for apolipoprotein B involves a cytidine to uridine transition at nucleotide 6666 and enables translation of two protein variants. The development of in vitro editing systems has led to the identification of three sequence requirements in this process. The mechanism for C→U editing requires specific sequences for editing site recognition, positioning of the catalytic activity and enhancement of catalytic efficiency. The dependence of in vitro editing on extract proteins has focused future research in this field on the identification of factors involved in apoB mRNA editing and the role of these factors in the assembly of ribonucleoprotein editosomes.     

Apolipoprotein B mRNA editing: a new tier for the control of gene expression.

Two forms of apolipoprotein (apo) B are found in mammals. The shorter form is translated from an edited mRNA in which a specific cytidine base is deaminated to a uridine, creating a new stop codon. Apo B mRNA editing is mediated by a site-specific cytidine deaminase that recognizes a downstream target sequence in the RNA. The enzyme has no energy or cofactor requirements and no RNA component, and thus bears no obvious relationship to RNA processing events such as splicing or polyadenylation. While apo B mRNA editing activity may have arrived late in evolution to target dietary lipid to the liver in mammals, the discovery of the editing activity in tissues and cells that do not express apo B suggests a more widespread role in the generation of RNA and protein diversity.     

An additional editing site is present in apolipoprotein B mRNA.

Human intestinal apolipoprotein (apo) B mRNA undergoes a C to U RNA editing at nucleotide 6666 to generate a translation stop at codon 2153, which defines the carboxy-terminal of apo B48. Here we show that two of eleven human intestinal cDNAs spanning residue 6666 were edited from a genomically-encoded C to a T at residue 6802 as well as at residue 6666. This additional editing converts Thr (ACA) codon 2198 to Ile (AUA). Synthetic RNA including the nucleotide 6802 was edited in vitro by intestinal extracts at 10-15% of the editing efficiency of nucleotide 6666. A sequence is identified as important for recognition by the editing activity. No secondary structural homology was identified between the two edited sites. No other sequence in the region between 6411 and 6893 nucleotides of apo B mRNA was found to be edited in vivo or in vitro. Apo B RNA editing extracts from intestine did not edit maize cytochrome oxidase II mRNA.     

Apolipoprotein B mRNA editing. Direct determination of the edited base and occurrence in non-apolipoprotein B-producing cell lines

In humans, apolipoprotein (apo) B48 is synthesized in the intestine as an obligatory constituent of chylomicrons. Apolipoprotein B48 is identical to the amino-terminal 2152 amino acids (240 kDa) of apoB100 and is translated from an edited apoB mRNA in which codon 2153 has been converted from glutamine (CAA) to what is recognized as a premature stop codon (UAA). To determine whether the apoB mRNA editing in fact converts cytosine 6666 in codon 2153 to uracil, we incubated a synthetic apoB RNA containing 32P-labeled cytosines in an in vitro editing system prepared from rabbit enterocytes. The in vitro edited RNA was purified and digested to nucleoside 5'-monophosphates, which were analyzed on two-dimensional thin-layer chromatography. We found that the edited base co-migrated with authentic uridine 5'-monophosphate. Thus, cytosine 6666 is converted to uracil, most likely by a nucleotide-specific cytosine deaminase. To determine whether apoB mRNA editing occurs in cell lines that do not synthesize apoB, we stably transfected a high expression vector containing 354 base pairs of apoB sequence into 18 different cell lines. We found apoB mRNA editing activity in five osteosarcoma cell lines and one epidermoid cell line, none of which synthesizes any detectable apoB. Thus, apoB mRNA editing occurs in cell lines that do not synthesize apoB, which suggests that mRNA editing may be a common biological phenomenon in eukaryotic cells.     

Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA.

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.