The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense 8-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sensestrand globin mRNA.Earlier, we reported the detection and partial characterization of antisense globin RNA in murine erythroleukemia (MEL) cells (1). This RNA appeared to be a complement of the corresponding sense globin mRNA. Indeed, antisense globin RNA had an electrophoretic mobility similar to that of globin mRNA, and it hybridized with probes corresponding to the 5'-and 3'-terminal segments of globin mRNA, as well as with probes corresponding to the entire globin mRNA. Experiments with whole cells (1) and with cytoplasts obtained by enucleation of MEL cells (2) indicated that antisense globin RNA, as well as its sense counterpart, is synthesized in the cytoplasm and suggested that it may serve as an intermediate in cytoplasmic globin mRNA synthesis by RNA-dependent RNA polymerase, an activity that has been detected in erythroleukemia cells (1), as well as in normal reticulocytes (3). If such a process indeed occurred physiologically, one would expect (i) to find the antisense globin RNA molecule not only in MEL cells but also in animal erythroid tissues, and (ii) that these molecules are precise complements of their sense counterparts. Accordingly, the present study was undertaken to test for the occurrence of antisense globin RNA in erythroid cells from mouse tissues and to analyze its primary structure, The publication costs of this article were defrayed in part by page charge...

Generation of double-stranded cDNA during reverse transcription of a variety of mRNA molecules is well known to involve the formation of covalently linked antisense and sense strands in a hairpin configuration. In the present study we have examined the sequence of molecular events which occurs during cDNA synthesis from mouse beta globin mRNA, in particular the self-priming event that initiates synthesis of sense-strand DNA. Upon completion of reverse transcription of globin mRNA and the removal of RNA template by RNase H activity associated with reverse transcriptase, the 3' end of cDNA snaps back to form a stable double-stranded structure, which is extended by reverse transcriptase to generate the sense DNA strand. Surprisingly, the fourteen 3' terminal nucleotides of the beta globin antisense DNA strand (cDNA) have strong complementarity with an internal segment of the same molecule corresponding to a portion of the 5'-untranslated region of the mRNA located just upstream of the translation start site. Efficient second strand cDNA synthesis appears to require the occurrence within the cDNA molecule of these two complementary elements, one of which must be 3'-terminal. A second surprising feature is that the strong complementarity between the terminal and the internal portions of the molecule exists in the antisense DNA and not in the sense mRNA strand. This is because A:C mismatches on the sense strand correspond to relatively stable T:G base pairs on the antisense strand. Such an extended region of complementarity within the segment of cDNA corresponding to the short 5' untranslated region of beta globin mRNA is unlikely to occur purely by chance, suggesting some underlying function. In this regard it is of interest that cDNAs of adult beta globin mRNAs from other mammalian species show a very similar arrangement of complementary elements, and that complementarity is heavily conserved, even when there are substitutions in nucleotide sequence.

Ligation-mediated RNA amplification was developed as a tool for analysis and determination of the termini of RNA molecules [Volloch et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10671-10675]. In this approach, T4 RNA ligase is used to join cellular RNA with a defined ribo-oligonucleotide. Although several additional enzymatic steps are involved in this type of analysis, the reliability of the entire procedure is determined by the initial ligation step, which marks and preserves the termini of cellular RNA molecules. We applied this approach to the analysis of the 5' terminus of beta globin mRNA in various murine erythroid cells. As expected, we detected RNA molecules with 5' ends terminating at the regular cap site as well as globin RNA molecules truncated at the 5' end. Unexpectedly, we also detected a class of beta globin mRNA which is identical to regular beta globin mRNA in every respect but contains 17, 29, or 31 additional nucleotides 5' to the regular cap site. These extensions correspond precisely to the genomic segments just upstream of the regular cap site and are probably generated by initiation of transcription of the globin gene upstream from the regular cap site. It is likely that the extended globin RNA is transcribed not from the TATA promoter, which regulates the transcription of regular murine globin mRNA, but from the GATA regulatory element located 30 nucleotides upstream of the 31-nucleotide extension, in a position identical to that of the active GATA promoter of the TATA-less chicken beta globin gene. The evolutionary conservation of this relationship suggests the importance of the GATA promoter element of the mouse beta globin gene and its possible involvement in developmental regulation of expression of this gene.

The possible transcription of the 5'-terminal cap nucleotide of mRNA by RNA-dependent DNA polymerase was examined by a single-step assay, based on the generation of a hairpin structure during reverse transcription of globin mRNA. Using this approach, we demonstrated that the 5'-terminal cap nucleotide of mRNA is indeed transcribed by RNA-dependent DNA polymerase into a 3'-terminal residue of cDNA and were able to measure the extent of such transcription. The observed transcription of the cap nucleotide raises a number of questions that may be addressed using the relatively simple single-step assay employed in the present study. Cap nucleotide transcription by RNA-dependent DNA polymerase may have important implications for our understanding of the mechanism of action of reverse transcriptases. It may represent a selection mechanism for only partial transcription of the 5' repeat element of viral RNA genome, thus generating a RNA fragment that may play a role in priming of second (plus) DNA strand during retroviral reverse transcription. Moreover, the demonstrated ability of complementary nucleotides to form hydrogen bonds, even when in a parallel orientation, may have interesting and important consequences.

A substantial amount of cytochrome oxidase subunit HI (COHI) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand corn RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand corn RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited corn RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of con RNA. The presence of negative-strand complements of coM RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.RNA metabolism in trypanosomes has a number of unusual aspects. Two novel reactions were first discovered in these organisms-trans-splicing and RNA editing. Trans-splicing, the processing of pre-mRNA by joining of independent RNA transcripts, accounts for the presence of an identical 39-nucleotide sequence at the 5' ends of all nuclear mRNAs in trypanosomes where the regular, cis-splicing is not observed (1, 2). RNA editing, which occurs in kinetoplast mitochondria, involves addition or occasionally the deletion of uridylate residues (3-7). This process occurs posttranscriptionally; and as a result the nucleotide sequence of the mature edited mRNA differs from that of the gene from which it is transcribed (8, 9).In the course of studies of RNA editing, we observed that a significant proportion of some edited mRNA species in Trypanosoma brucei is the product of an actinomycin D-resistant (i.e., presumably DNA-independent) transcriptional process (10). This finding suggested the occurrence in T. brucei of yet a third unusual feature of RNA metabolism, RNA-dependent RNA synthesis via negative-strand RNA intermediates. Here we describe the identification and characterization of negative-strand complements for cytochrome oxidase subunit III (COIII) RNA in procyclic T. brucei. MATERIALS AND METHODSCell Culture. The procyclic form of clone 570 of T. brucei, stock EVE10 (from I. Cunningham, University of Massachusetts, Amherst), was grown in Dulbecco's modified Eagle's medium with 20% fetal bovine serum.Isolation of RNA. Cells were lysed at 0C in 20 mM Hepes, pH 8.2/360 mM NaCl/4 mM MgCl2/8% (wt/vol) sucrose/ 1.5% (vol/vol) Triton X-100 containing RNasin (Promega) at 1000 units/ml. The cytoplasmic fraction was treated with proteinase K and extracted with phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol).RNA Electrophoresis, Blotting, and Hybridization. RNA samples were denatured and subjected to electrophoresis in 1.5% agaro...

RNA editing, a novel and unexpected type of information processing, was first demonstrated in the kinetoplasts of certain protozoans. It is a remarkable phenomenon: certain species of messenger RNA have nucleotide sequences that differ greatly from the sequences of the genes from which they are presumably transcribed. The differences are usually due to addition of uridylate residues, but occasionally also to their deletion. The most spectacular case of editing known occurs in the mRNA of the mitochondrial gene for subunit III of cytochrome oxidase (COIII) in Trypanosoma brucei. This mRNA is twice the length of its gene owing to the addition of several hundred uridylate residues and a few deletions, spread over the entire length of the RNA molecule. Whereas unedited RNA molecules, the nucleotide sequences of which correspond to the genomic sequence, have been isolated, no DNA template corresponding to the edited RNA sequence has been detected in either the mitochondrial or nuclear genome. It was suggested, therefore, that unedited mRNAs are transcribed from mitochondrial DNA and then edited post-transcriptionally by endonucleolytic cleavage of the primary transcript at specific sites, followed by insertion or deletion of uridylate residues and religation. We have now examined the general nature of the RNA editing process to determine whether it involves the insertion of nucleotide residues into pre-existing molecules or the continuous de novo synthesis of edited mRNA. The results of our experiments rule out an insertion mechanism and strongly indicate that edited mRNA is synthesized as a unit.

Abstract. In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212 we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive-and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive-and negativestrand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg +÷, was inhibited by Mn ÷+, and was unaffected by the addition of Zn +÷. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and a-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNAdependent RNA synthesis in differentiated ceils and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.

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