An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

Synthesis of the pol gene products of most retroviruses requires ribosomes to shift frame once or twice in the -1 direction while translating gag-pol mRNA. The viral signals for frameshifting include a heptanucleotide sequence on which the shift occurs and higher-order RNA structure just downstream of the shift site. We have made site-directed mutations in two stems (S1 and S2) of a putative RNA pseudoknot that begins 7 nucleotides 3' of the previously identified shift site (A AAA AAC) in the gag-pro region of mouse mammary tumor virus (MMTV) RNA. The mutants confirm the predicted structure, show that loss of either S1 or S2 impairs frameshifting, and exclude alternative RNA structures as significant for frameshifting. The importance of the MMTV pseudoknot has been further demonstrated by showing that shift sites from two other retroviruses function more efficiently in the position of the MMTV site than in their native contexts. However, the MMTV pseudoknot cannot promote detectable frameshifting in the absence of a recognizable upstream shift site. In addition, the species of tRNA that reads the second codon in the shift site appears to be a critical determinant, since changing the 7th nucleotide in the MMTV gag-pro shift site from C to A, U, or G severely impairs frameshifting.     

Amplifiable messenger RNA.

RNA molecules were prepared that consisted of an mRNA encoding chloramphenicol acetyltransferase embedded within the sequence of midivariant RNA, which is a template for the RNA-directed RNA polymerase Q beta replicase. These recombinant RNAs were shown to be bifunctional: they are amplified exponentially by incubation with Q beta replicase, and the replicated RNA serves as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase. The availability of amplifiable mRNAs will enable relatively large amounts of protein to be synthesized in vitro.     

Mouse thymidylate synthase messenger RNA lacks a 3'' untranslated region.

Analysis of the sequence of cDNA corresponding to mouse thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) mRNA revealed that the termination codon TAA was followed immediately by a poly(A) sequence. This raised the possibility that mouse thymidylate synthase mRNA lacks a 3' untranslated region. In the present study, we have further investigated this possibility. DNA corresponding to the 3' end of the thymidylate synthase gene was isolated from a genomic library. The sequence of the genomic DNA was identical to that of the cDNA in the coding region. However, the termination codon was TAG in the genomic sequence rather than TAA, and poly(A) was not present in the genomic DNA. Sequences flanking the site of poly(A) addition were in good agreement with polyadenylylation consensus sequences. S1 nuclease analysis revealed that approximately 80% of the thymidylate synthase mRNA molecules were polyadenylylated at the termination codon. A secondary polyadenylylation site was detected 190-200 nucleotides downstream of the primary site. We conclude that the major species of mouse thymidylate synthase mRNA lacks a 3' untranslated region and that the final A of the termination codon is added by poly(A) polymerase. It appears that a 3' untranslated region is not essential for the accumulation or translation of this mRNA.     

Kallikrein messenger RNA in rat arteries and veins.

Glandular kallikrein (EC 3.4.21.8) belongs to a subgroup of serine proteases coded by a multigene family. A kininogenase resembling glandular kallikrein has been identified in vascular tissue; however, it is not clear whether it is synthesized by vascular tissue or taken up from plasma. To determine the potential for kallikrein synthesis in vascular tissues, we tested whether messenger RNA (mRNA) for glandular kallikrein is present in rat arteries and veins. Poly(A+) RNA was isolated from pools of arteries or veins (n = 3, 30 rats each). Poly(A+) RNA from the kidney and liver was used as a positive and negative control, respectively. As a probe, we used rat pancreatic kallikrein 32P-labeled complementary DNA, which recognizes mRNA of the entire rat kallikrein family. Slot-blot analysis indicated that kallikrein mRNA was present in mRNA from the arteries, veins, and kidney but not from the liver. Poly(A+) RNA from arteries and veins contained approximately 1% as much kallikrein mRNA as that from the kidney. To confirm the slot-blot results and determine whether the mRNA for true glandular kallikrein was present in vascular tissue, we employed a polymerase chain reaction assay, first using primers specific for the entire kallikrein family (which amplify a 430-bp fragment) and then using primers specific for true glandular kallikrein mRNA (which amplify a 370-bp fragment). After the polymerase chain reaction assay, both arteries and veins showed fragments of these sizes when tested with rat kallikrein complementary DNA probe, thus confirming the presence of glandular kallikrein mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short-lived methylated messenger RNA in mouse kidney.

In experiments originally designed to examine selective turnover of methylated "caps" in renal mRNA, we observed that [3H]methyl label decayed from mRNA containing poly(A) with a half-life of 1-2 hr. (Caps are blocked, methylated mRNA sequences of the general structure m7GpppNm p(1 or 2)Np.). To distinguish between metabolism of short-lived mRNA and discriminate turnover of "caps", we compared residual [3H]methyl label in 5' and 3'mRNA fragments prepared from mRNA isolated during the decay period. Hydrolysis of mRNA at 0 degrees with dilute KOH before oligo(dT)-cellulose selection produced 5' mRNA fragments enriched with an alkali-resistant oligonucleotide with a -5 charge; the 3' mRNA fraction was correspondingly reduced in oligonucleotide content. Since methyl label disappeared at the same rate from both fractions, we conclude that mouse kidney contains short-lived mRNA and that the "caps" of these labile mRNAs turn over with the rest of the mRNA molecule.     

An evaluation of messenger RNA competition in the shutoff of human interferon production.

The process that shuts off poly(I)-poly(C)-induced interferon production in a strain of diploid human fibroblasts (FS-4)-involves the synthesis of new RNA, presumably nuclear heterogeneous RNA. When cultures in the shutoff phase of interferon production are treated with actinomycin D (5 mug/ml) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB 40 muM), the rate of interferon production continues to decline for a further 3-4 hr, but then tends to level off. The treated cultures maintain interferon production at a reduced level for at least 10 hr. The residual rate of interferon production is higher in cultures which received actinomycin D or DRB early in the shutoff phase as compared to the rate in cultures treated late.     

An autoregulatory region in protein kinase C: the pseudoanchoring site.

We have previously identified receptors for activated C kinase (RACKs) as components of protein kinase C (PKC) signaling. RACK1, a recently cloned 36-kDa RACK, has short sequences of homology to PKC. A possible explanation for the homologous sequences between the ligand (PKC) and its intracellular receptor (RACK1) may be that, similar to the pseudosubstrate autoregulatory sequence on PKC, there is also a pseudo-RACK1 binding site on the enzyme. If this is the case, peptides with these sequences (derived from either RACK1 or PKC) are expected to affect PKC binding to RACK1 in vitro and PKC-mediated functions in vivo. Here, we show that the PKC-derived peptide (pseudo-RACK1 peptide), but not its RACK1 homologue, modulated PKC function both in vitro and in vivo. Our data suggest that the pseudo-RACK1 peptide binds and activates PKC in the absence of PKC activators and thereby acts as an agonist of PKC function in vivo. Therefore, the pseudo-RACK1 sequence in PKC appears to be another autoregulatory site; when PKC is in an inactive conformation, the pseudo-RACK1 site interacts with the RACK-binding site. Activation of PKC exposes the RACK-binding site, enabling the association of the enzyme with its anchoring RACK. Similar pseudoanchoring sites may regulate the function of other protein kinases.     

Nucleotide sequences of the 3'-terminal untranslated region of messenger RNA for human beta globin chain.

In normal messenger RNA for the human beta-globin chain, nucleotide sequences have been identified which can be matched to the amino-acid sequence of the abnormally long segment of the beta-chain of hemoglobin Cranston. The finding of these sequences strengthens the hypothesis that the betaCranston chain arose by a frameshift mutation allowing the "readthrough" of the normal termination codon and translation of usually untranslated portions of the messenger RNA for the beta-globin chain. The oligonucleotides which match the amino-acid sequence of hemoglobin Cranston provide a sequence of 36 nucleotides which follows the normal beta-chain termination codon UAA.     

Androgen-dependent angiotensinogen and renin messenger RNA expression in hypertensive rats.

Our previous studies demonstrated that the sexually dimorphic pattern of hypertension in the spontaneously hypertensive rat is androgen dependent. Gonadectomy retards the development of hypertension in young males, but not in females, and administration of testosterone propionate to gonadectomized spontaneously hypertensive rats of both sexes confers a male pattern of blood pressure development. The current study tested the hypothesis that renal and hepatic renin and angiotensinogen gene expression are also androgen dependent in the spontaneously hypertensive rat. Male and female spontaneously hypertensive rats underwent gonadectomy or a sham operation at 4 weeks of age. Subgroups of gonadectomized rats of both sexes were implanted with a 15-mm or 30-mm Silastic capsule filled with testosterone at the same time the gonadectomy was performed; a third group received an empty Silastic capsule. Northern and slot blot analyses were used to characterize and quantitate renin and angiotensinogen messenger RNA (mRNA) in the kidney and liver 18 weeks after the gonadectomy. Blood pressure, plasma renin activity, and hepatic angiotensinogen mRNA levels were higher in intact males than in females. Orchidectomy retarded the development of hypertension and lowered plasma renin and renal and hepatic angiotensinogen mRNA levels, and testosterone replacement restored the male pattern of hypertension and plasma renin and increased renal and hepatic angiotensinogen mRNA. Ovariectomy did not alter blood pressure or plasma renin but did lower renal renin and renal and hepatic angiotensinogen mRNA; testosterone increased blood pressure, plasma renin, renal renin and angiotensinogen mRNA, and hepatic angiotensinogen mRNA levels in ovariectomized females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine messenger RNA stability is enhanced in tumor cells.

Hematopoietic growth factors are produced by a number of human tumors. We extracted RNA from selected human tumor cells known to produce at least one hematopoietic growth factor and found high levels of abnormally stable cytokine messenger (m)RNA. Half-life experiments performed after preventing RNA synthesis by exposing cells to actinomycin D before RNA extraction showed stabilization of cytokine messages in tumor cells in liquid culture as well as in human tumor xenografts grown in mice. Exposure to the phorbol ester phorbol 12-myristate 13-acetate (TPA) caused enhancement of granulocyte-macrophage colony-stimulating factor (GM-CSF) message level in lung cancer cells and in control fibroblasts but elevated levels persisted far longer in the tumor cells. In normal cells, an AU-rich sequence in the 3' untranslated region of cytokine mRNAs confers lability to the message. Although a beta-globin gene expression vector containing this region appears to produce unstable mRNA in lung cancer cells, cytokine mRNAs, which also contain this sequence, are very stable in the tumors we studied. This may indicate that another region of the cytokine mRNA molecule is of greater importance than the AU-rich region in determining mRNA stability in tumor cells.     

Mapping of adenovirus messenger RNA by electron microscopy.

Late adenovirus messenger RNA was annealed to complementary regions of partially melted viral double-stranded DNA. The RNA. DNA hybrid regions within the DNA molecules were visualized as loops in the electron microscope. Loops occurred at several regions of the DNA, most frequently, however, at a location near the center of the molecule. This hybridization technique appears well suited for an accurate mapping of messenger RNA, as well as for studies of RNA processing.