A characterization of globin mRNA sequences in the nucleus of duck immature red blood cells

Studies were performed with duck immature red blood cells to identify and characterize the globin mRNA sequences in nuclear RNA. Annealing of ³H-globin cDNA to unlabeled nuclear RNA has identified three distinct size classes of nuclear RNA molecules containing globin mRNA sequences. The largest size class contained 1–2% of total nuclear globin mRNA sequences and sedimented through 85% formamide-sucrose gradients at the same rate as 28S ribosomal RNA. Chromatography on oligo(dT)-cellulose indicated that most of these molecules are not polyadenylated. The bulk of nuclear globin mRNA sequences (70%) was contained in polyadenylated RNA molecules which sedimented at 16.5S. The remainder of nuclear globin mRNA sequences (～30%) was detected in molecules sedimenting at 10S (the position of cytoplasmic globin mRNA).To determine whether a precursor-product relationship exists between these nuclear molecules and cytoplasmic globin mRNA, pulse-label and chase experiments were performed. Labeled globin mRNA sequences were assayed by annealing to globin cDNA-cellulose. Labeled 28S nuclear globin RNA sequences could not be detected, perhaps due to technical reasons. 16.5S nuclear globin RNA was labeled and chased into cytoplasmic globin mRNA sequences. The half-life of 16.5S nuclear globin RNA was estimated to be less than 30 min. These results demonstrate that in duck immature red blood cells, globin mRNA is transcribed as a larger precursor. Furthermore, size characterization of this precursor during pulse-label and chase periods suggests that it is processed within the nucleus to 10S globin RNA.     

Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.

The proteins that interact with cytoplasmic and nuclear polyadenylated RNA in adenovirus type 5 (Ad5) infection of HeLa cells were examined by UV-induced RNA-protein cross-linking in intact cells. The Ad5 100-kilodalton late nonvirion protein (100K protein) was cross-linked to both host and viral polyadenylated cytoplasmic RNA (mRNA). The cross-linking of the 100K protein to mRNA appears to correlate with productive infection, because the protein is not cross-linked to mRNA in abortive infection of wild-type Ad5 in monkey cells (CV-1) even though normal amounts of it are produced. However, when CV-1 cells are infected with Ad5 hr404, and Ad5 mutant which overcomes the host restriction to wild-type Ad5 infection in these cells, the 100K protein is cross-linked to mRNA. To identify and obtain antibodies to RNA-contacting proteins, a mouse was immunized with oligo(dT)-selected cross-linked RNA-protein complexes from Ad5-infected cells and the serum was used for immunoblotting experiments. It was found that in addition to the 100K protein, the Ad5 72K DNA-binding protein is also associated with RNA in the infected cells. The 72K DNA-binding protein is cross-linked to polyadenylated nuclear RNA sequences. These findings indicate that adenovirus proteins interact with RNAs in the infected cell and suggest possible mechanisms for the effects of the virus on mRNA metabolism.     

Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus.

The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.     

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).     

4,5-S-RNAI in virus-specific polyribosomes from ascitic Krebs 2 carcinoma cells infected with encephalomyocarditis virus

Polyribosomes of Krebs 2 ascite carcinoma cells non-infected and infected with encephalomyocarditis (EMC) virus contain a heterogeneous population of low molecular weight small RNAs. Analysis of the RNAs by polyacrylamide gel electrophoresis did not reveal any qualitative differences in the small RNA sets within the composition of polyribosomes from virus-infected and non-infected cells. However, the content of one of the small RNAs was markedly elevated in polyribosomes from virus-infected cells. As can be followed from partial determination of its primary structure, this small mRNA is identical to 4,5S-RNAI previously detected in the nuclei of Novikov hepatoma cells of the rat. The data obtained suggest that 4,5S-RNAI can be involved in the regulation of protein synthesis in virus-infected cells.     

UTP-dependent turnover of Trypanosoma brucei mitochondrial mRNA requires UTP polymerization and involves the RET1 TUTase

Trypanosoma brucei mitochondria possess a unique RNA decay pathway in which rapid degradation of polyadenylated mRNAs is dependent on the addition of UTP, as measured by in organello pulse chase assays. To determine the mechanism by which UTP stimulates the degradation of polyadenylated RNAs, we performed in organello pulse chase assays under different conditions. Treatment of mitochondria with proteinase K revealed that UTP does not act through a receptor on the surface of the mitochondria. To determine if the UTP-stimulated RNA decay pathway is triggered by the mitochondrial energy state or ATP:UTP ratio, increasing ATP was added to a constant amount of UTP during the chase period of the assay. Results indicate that rapid turnover is responsive to UTP and not the ATP:UTP ratio. Experiments using UTP analogs demonstrate that UTP polymerization into RNAs is necessary for UTP-dependent degradation. Furthermore, experiments performed with RNAi cells indicate that the RET1 terminal uridylyl transferase (TUTase) is required for UTP-dependent decay of polyadenylated RNAs. Overall, these results show that degradation of polyadenylated RNAs in T. brucei mitochondria can occur through a unique mechanism that requires the polymerization of UTP into RNAs, presumably by the RET1 TUTase.     

Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs

Polyadenylation of RNAs plays a critical role in modulating rates of RNA turnover and ultimately in controlling gene expression in all systems examined to date. In mitochondria, the precise mechanisms by which RNAs are degraded, including the role of polyadenylation, are not well understood. Our previous in organello pulse-chase experiments suggest that poly(A) tails stimulate degradation of mRNAs in the mitochondria of the protozoan parasite Trypanosoma brucei (Militello, K. T., and Read, L. K. (2000) Mol. Cell. Biol. 21, 731-742). In this report, we developed an in vitro assay to directly examine the effects of specific 3'-sequences on RNA degradation. We found that a salt-extracted mitochondrial membrane fraction preferentially degraded polyadenylated mitochondrially and non-mitochondrially encoded RNAs over their non-adenylated counterparts. A poly(A) tail as short as 5 nucleotides was sufficient to stimulate rapid degradation, although an in vivo tail length of 20 adenosines supported the most rapid decay. A poly(U) extension did not promote rapid RNA degradation, and RNA turnover was slowed by the addition of uridine residues to the poly(A) tail. To stimulate degradation, the poly(A) element must be located at the 3' terminus of the RNA. Finally, we demonstrate that degradation of polyadenylated RNAs occurs in the 3' to 5' direction through the action of a hydrolytic exonuclease. These experiments demonstrate that the poly(A) tail can act as a cis-acting element to facilitate degradation of T. brucei mitochondrial mRNAs.     

Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.     

Structural analysis of the intracellular RNAs of murine mammary tumor virus.

We have characterized murine mammary tumor virus (MuMTV)-specific RNA in several types of cells in which viral DNA is transcribed into RNA: cultured GR mouse mammary tumor cells, S49 lymphoma cells from BALB/c mice, lactating mammary glands from C57BL/6 mice, and mink lung cells infected in vitro with MuMTV. In all cell types studied, there are three distinct species of intracellular viral RNA, with sedimentation coefficients of 35S, 24S, and 13S (or molecular weights of 3.1 X 10(6), 1.5 X 10(6), and 0.37 X 10(6), as determined by rate-zonal sedimentation in sucrose gradients and by electrophoresis in agarose gels under denaturing conditions. These three viral RNA species appear to be present regardless of viral RNA concentration, responsiveness to glucocorticoid hormones, production of extracellular virus, and use of either endogenous or acquired MuMTV proviral DNA as template. The three viral RNAs display characteristics of mRNAs in that they are polyadenylated, associated with polyribosomes, and released from polyribosomes by treatment with EDTA; hence all three species presumably direct the synthesis of virus-coded proteins. The two larger species of viral RNA are probably responsible for synthesis of the structural proteins of the virion, but the function of the 13S RNA is not known. Both of the subgenomic RNAs contain sequences found at the 3' terminus of 35S (or genomic) RNA. However, only the 24S RNA (not the 13S RNA) contains sequences which are located at the 5' terminus of 35S RNA and are apparently transposed during RNA synthesis of maturation, as described for subgenomic mRNA's of other retroviruses.     

Subcellular localization of RNAs in transfected cells: role of sequences at the 5' terminus

We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported.     

Protein synthesis in BHK-21 cells infected with semliki forest virus.

[3H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [3H-A1leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating robosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action

In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNA-directed mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein. Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.     

Translational capacity of deadenylated messenger RNA

In order to determine whether the functional capacity of polyadenylated messenger RNA is altered when the poly(A) segment is shortened as a consequence of mRNA aging, we compared the abilities of differentially labeled new and old mRNAs to engage ribosomes. Experiments were performed using both exponentially growing mouse L cells and L cells in which the polyribosomes were temporarily disassembled as a result of a temperature shock. Our results indicate that new and old messages engage ribosomes with roughly equivalent efficiencies, suggesting, therefore, that length of poly(A) is not a determining factor for mRNA translation. We then examined the question of whether an mRNA that was originally polyadenylated could function if the Poly(A) segment were removed. This was accomplished by submitting polyadenylated mRNA to a limited digestion with a 3′-OH specific exoribonuclease, reisolating the residual polyadenylated and deadenylated mRNA, and measuring their template activities using a cell-free protein synthesizing system prepared from wheat embryos. Such experiments also failed to reveal any effect of the poly(A) on the translational capacity of the mRNA.     

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.