Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing.

Nematode trans-spliced leader (SL) RNAs are composed of two domains, an exon [the 22-nucleotide spliced leader] and a small nuclear RNA (snRNA)-like sequence. Participation in vitro of the spliced leader RNA in trans-splicing reactions is independent of the exon sequence or size and instead depends on features contained in the snRNA-like domain of the molecule. Chemical modification interference analysis has revealed that two short sequence elements in the snRNA-like domain are necessary for SL RNA activity. These elements are sufficient for such activity because when added to a 72-nucleotide fragment of a nematode U1 snRNA, this hybrid RNA could participate in trans-splicing reactions in vitro. One of the critical sequence elements may function by base-pairing with U6 snRNA, an essential U snRNA for both cis- and trans-splicing.

Translational efficiency of cMyc mRNA in Burkitt lymphoma cells.

The translational efficiency of cMyc mRNAs was assessed in a variety of cell lines: HeLa cells and Epstein-Barr virus-transformed lymphocytes, both of which contain only the germ line cMyc allele; Daudi, a Burkitt cell line containing a translocated cMyc gene with no apparent alteration; and P3HR-1, a Burkitt line in which the 5' end of the translocated cMyc gene has been altered by the chromosomal translocation. Translational efficiency was inferred by measuring the number of ribosomes associated with the cMyc mRNA, using a procedure by which individual polysomal fractions were analyzed by blot hybridization. Since polysome size is a function of the length of the translated sequence as well as the rate of initiation of protein synthesis, we also determined the number of ribosomes associated with a control mRNA (alpha tubulin) which codes for a protein of similar size to cMyc. We found that the cMyc mRNA was associated with a number of ribosomes comparable to that associated with alpha tubulin mRNA in all the cell lines tested.

Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus.

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Heterogeneous nuclear RNA promotes synthesis of (2',5')oligoadenylate and is cleaved by the (2',5')oligoadenylate-activated endoribonuclease.

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.

c-erbB activation in avian leukosis virus-induced erythroblastosis: multiple epidermal growth factor receptor mRNAs are generated by alternative RNA processing.

Avian leukosis virus-induced erythroblastosis results from the specific interruption of the host oncogene, c-erbB, by the insertion of an intact provirus. This insertion results in the expression of two size classes (3.6 and 7.0 kilobases [kb]) of truncated c-erbB transcripts which are initiated in the 5' long terminal repeat of the integrated provirus. Through sequence analysis of erbB cDNA clones we have previously shown that the 3.6-kb activated erbB mRNA contains portions of viral gag and env genes fused to c-erbB sequences (T.W. Nilsen, P.A. Maroney, R.G. Goodwin, F.M. Rottman, L.B. Crittenden, M.A. Raines, and H.-J. Kung, Cell 41:719-726, 1985). In this report we show that the 7-kb mRNA differs from the shorter activated c-erbB mRNA in the length of its 3' untranslated sequence such that the longer mRNA has an extremely long (4.3 kb) 3' untranslated sequence. Additionally, we demonstrate that activated c-erbB mRNA precursors can be processed by alternative splicing to yield mRNAs with viral gag sequences fused directly to c-erbB sequences. Finally, blot hybridization evidence suggests that the two size classes of activated c-erbB mRNA in erythroblastic tissue represent truncated versions of the two c-erbB mRNAs present in normal tissue.

Unequal distribution of N6-methyladenosine in influenza virus mRNAs.

Influenza virus mRNA is posttranscriptionally methylated at internal adenosine residues to form N6-methyladenosine (m6A). It has been previously shown that there is an average of three m6A residues per influenza virus mRNA (R. M. Krug, M. A. Morgan, and A. J. Shatkin, J. Virol. 20:45-53, 1976). To determine the distribution of m6A in the different influenza virus mRNAs, we purified six of the mRNAs by hybrid selection, digested them with nuclease, and determined their methylation patterns by high-pressure liquid chromatography. The amount of m6A in the different mRNAs varied from one in matrix to eight in hemagglutinin.

Accurate processing of human pre-rRNA in vitro.

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

Accurate and efficient RNA polymerase III transcription in a cell-free extract prepared from Ascaris suum embryos.

High-speed supernatant (S100) extracts derived from homogenized Ascaris suum embryos efficiently transcribe added RNA polymerase III templates including cloned 5S rRNA genes of the filarial parasite Brugia malayi. Several criteria, including two-dimensional RNase T1 oligonucleotide fingerprint analysis, indicate that in vitro transcription is accurately initiated and terminated.

MicroRNAs, mRNAs, and translation.

MicroRNAs (miRNAs) comprise a large family of regulatory molecules that repress protein production from targeted mRNAs. Although it is now clear that miRNAs exert pervasive effects on gene expression in animal cells, the mechanism(s) by which they function remains poorly understood. We have analyzed the subcellular distribution of miRNAs in actively growing HeLa cells and find that the vast majority are associated with actively translating mRNAs in polysomes. We also find that a specific miRNA-regulated mRNA (KRAS) is polysome associated and that its translation is impaired, apparently at the level of elongation. These observations are discussed in light of our current understanding of mechanism of miRNA function.

Inhibition of double-stranded ribonucleic acid activated protein kinase and 2',5'-oligo(adenylic acid) polymerase by ethidium bromide.

The activation of two enzymes induced by interferon, a protein kinase and the 2',5'-oligo(adenylic acid) polymerase [2'5'-oligo(A) polymerase], is inhibited by ethidium bromide. The activation of these enzymes requires double-stranded RNA (dsRNA), and binding of ethidium to dsRNA inhibits the activation process. This was shown by determining the concentration of ethidium inhibitory for poly(A) . poly(U)- and poly(I) . poly(C)-activated reactions. Activation of both protein kinase and 2',5'-oligo(A) polymerase is inhibited by much lower concentrations of ethidium with the former polymer as activator than with the latter polymer. Correspondingly, in the presence of magnesium, ethidium binds with much greater affinity to poly(A) . poly(U) than to poly(I) . poly(C). Synthesis of 2',5'-oligo(A) with poly(A) . poly(U) as activator is arrested by adding low ethidium concentrations, but it is resumed upon addition of poly(I) . poly(C). Kinase activity, however, is not inhibited when ethidium is added after the activating dsRNA. This suggests that the kinase interacts with dsRNA in a manner different from the 2',5'-oligo(A) polymerase interaction.

Mechanisms of action of human interferons. Induction of 2'5'-oligo(A) polymerase.

Interferons induce an enzymatic activity that polymerizes ATP into 2'5'-oligoadenylate. This enzyme, designated 2'5'-oligo(A) polymerase, is induced in HeLa cells by type I interferon with a faster kinetics than by type II (immune) interferon. Cells treated with type I interferon in the presence of cycloheximide show elevated levels of polymerase when the inhibitor or protein synthesis is removed and actinomycin D is added to the cultures to prevent further synthesis of mRNA. This suggests that, in the presence of cycloheximide, interferon can activate transcription of mRNA for the 2'5'-oligo(A) polymerase, which is subsequently translated upon removal of this inhibitor. In contrast, cells treated with type II interferon in the same way do not show increased levels of polymerase. This indicates that type II interferon cannot activate transcription of a stable mRNA for the polymerase when protein synthesis is inhibited. This observation and the different kinetics of induction of 2'5'-oligo(A) polymerase suggest major differences in the mechanism of action of the different types of interferon, with type I being a "direct" inducer of the polymerase and type II an "indirect" inducer. The commitment of HeLa cells to the induction of 2'5'-oligo(A) polymerase declines if the cells are exposed to type I interferon in the presence of an inhibitor of RNA synthesis. This suggests that transcription of specific mRNAs is transiently activated by interferon and that repeated interaction of interferon with its receptor may be required for prolonged transcription of the mRNA for 2'5'-oligo(A) polymerase.

U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro.

In nematodes, a fraction of mRNAs acquires a common 22-nucleotide 5'-terminal spliced leader sequence via a trans-splicing reaction. The same premessenger RNAs which receive the spliced leader are also processed by conventional cis-splicing. Whole cell extracts prepared from synchronous embryos of the parasitic nematode Ascaris lumbricoides catalyze both cis- and trans-splicing. We have used this cell-free system and oligodeoxynucleotide directed RNase H digestion to assess the U small nuclear RNA requirements for nematode cis- and trans-splicing. These experiments indicated that both cis- and trans-splicing require intact U2 and U4/U6 small nuclear ribonucleoproteins (snRNPs). However, whereas cis-splicing displays the expected requirement for an intact U1 snRNP, trans-splicing is unaffected when approximately 90% of U1 snRNP is degraded. These results suggest that 5' splice site identification differs in nematode cis- and trans-splicing.

Double-stranded RNA causes synthesis of 2',5'-oligo(A) and degradation of messenger RNA in interferon-treated cells.

Interferon-treated HeLa cells were incubated with [3H]uridine to label mRNA and were then exposed to the double-stranded RNA poly(inosinic acid).poly(cytidylic acid) (In.Cn). The incubation with In.Cn greatly enhanced the decay of mRNA. When the cells were incubated in this way in the presence of cycloheximide, which blocks ribosome movement along mRNA, extensive polysome degradation was detected in interferon-treated cells. Products of degradation of mRNA were recovered from monosomes which were presumably formed as a result of endonucleolytic breaks of mRNA. This endonucleolytic activity was correlated with the formation of 2',5'-oligo(A) by an enzyme induced by interferon and activated by double-stranded RNA; the 2',5'-oligo(A) was previously shown to activate an endonuclease in cell extracts. The 2',5'-oligo(A) levels in cells were measured by a competition-binding assay. Details of the procedure used are described, including synthesis of highly radioactive (2'-5')pppA3[32P]cytidine 3',5'-diphosphate, separation of 2',5'-oligo(A) binding from degrading activities, and specificity of the assay.

Inhibition of protein synthesis in reovirus-infected HeLa cells with elevated levels of interferon-induced protein kinase activity.

Protein synthesis was inhibited in one line of interferon-treated HeLa cells (line 2) upon infection with reovirus, but not in different HeLa cells (line 1) treated in the same way. The inhibition resulted in polysome runoff, suggesting that it was due to an impairment of peptide chain initiation. Interferon induces the synthesis of a protein kinase, which is activated in cell-free systems by double-stranded RNA and phosphorylates the alpha subunit of eukaryotic initiation factor 2, thus inhibiting the initiation of protein synthesis. Therefore, we measured the level of this protein kinase in extracts prepared from the two HeLa cell lines. Cells of line 2 showed about 3-4 times more protein kinase activity than cells of line 1. The inhibition of protein synthesis upon infection with reovirus was correlated with an increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in interferon-treated cells labeled with 32P. The kinase was presumably activated in intact cells by viral double-stranded RNA, but this activation resulted in inhibition of protein synthesis only in cells with elevated levels of the kinase.

Nuclease protection of RNAs containing site-specific labels: a rapid method for mapping RNA-protein interactions.

This report describes a method that combines nuclease protection and site-specific labeling to determine sites and extents of RNA-protein interactions. The utility of the method is demonstrated by the analysis of the binding of factors to the 3' splice site region of a pre-mRNA labeled at three specific positions. This "reverse footprinting" technique should be widely applicable to a variety of questions concerning RNA-protein interactions.

Synthesis of (2'-5')oligoadenylate and activation of an endoribonuclease in interferon-treated HeLa cells infected with reovirus.

Treatment with interferon protected HeLa cells from infection with reovirus. This virus apparently activated an antiviral mechanism that was detected by the presence of (2'-5')oligoadenylate [(2'-5')An] in intact cells. The (2'-5')An was previously shown to activate an endoribonuclease, RNase L. We measured (2'-5')An by a sensitive competition-binding assay in cells infected at different multiplicities and for different lengths of time. Nanomolar concentrations of (2'-5')An were detected in cells infected at a multiplicity of greater than 5 after 2 h of infection, the time at which the infecting virions were uncoated. The level of (2'-5')An increased up to 6 h postinfection but declined afterward. To establish whether viral mRNAs were cleaved by RNase L, we analyzed the RNA extracted from infected cells by a highly specific hybridization assay on Northern blots. Full-sized reovirus mRNAs were detected in control infected cells, but not in interferon-treated infected cells, at 6 h postinfection. At this time, a nuclease activity could be detected in these cells by demonstration of cleavage of rRNA, degradation of cellular mRNA, and polysome breakdown in the presence of emetine. Since this inhibitor freezes ribosomes, cleavage of mRNA between ribosomes could only be accounted for by an endonuclease, presumably RNase L.

Transcription and cap trimethylation of a nematode spliced leader RNA in a cell-free system.

Maturation of a fraction of mRNAs in nematodes involves the acquisition of a common 5' terminal spliced leader sequence derived from a nonpolyadenylylated spliced leader RNA by trans splicing. We have developed a cell-free system prepared from Ascaris lumbricoides embryos that accurately and efficiently synthesized the spliced leader RNA of A. lumbricoides. Transcription of the spliced leader RNA was catalyzed by RNA polymerase II, and the majority of the spliced leader RNAs synthesized in vitro possessed a trimethylguanosine cap structure identical to that found on in vivo-synthesized spliced leader RNA.

Functional reconstitution of U6 snRNA in nematode cis- and trans-splicing: U6 can serve as both a branch acceptor and a 5' exon.

Maturation of nuclear pre-mRNAs in nematodes requires both cis- and trans-splicing. Both processing pathways involve analogous two-step phosphotransfer reactions and both are dependent upon the integrity of U6 snRNA. We have developed a functional reconstitution assay to assess the U6 snRNA sequence requirements for cis- and trans-splicing. Branch formation between the splicing substrates and U6 snRNA was observed. The frequency of this event was greatly enhanced when a highly conserved sequence in U6 snRNA was altered by mutation. In cis- and trans-splicing reactions reconstituted with this mutant U6 snRNA the liberated exon of U6 proceeded through the second step of splicing using the appropriate splice acceptor sites. These results demonstrate covalent interactions between a U snRNA required for splicing and a splicing substrate, and they provide evidence for an unexpected degree of catalytic flexibility within the spliceosome.