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The chloroplast genome of a dinoflagellate consists of a group of small circular DNA molecules (minicircles), most of which carry a single gene. With RT-PCR, primer extension, and Northern analyses, we show that the entire minicircle is transcribed and that some minicircles can produce RNAs larger than themselves. Using an RNA ligase-mediated rapid amplification of cDNA ends method, we were able to detect large processed precursors that are generated by endonucleolytic cleavage of an even longer molecule. This cleavage produces the mature mRNA 3'-end and at the same time the 5'-end of the precursor. The tRNAs encoded by the petD and psbE minicircles appear to be processed in the same way. We propose a "rolling circle" model for chloroplast transcription in which transcription would proceed continuously around the minicircular DNA to produce transcripts larger than the minicircle itself. These transcripts would be further processed into discrete mature mRNAs and tRNAs.

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. We have demonstrated previously that ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3'-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions approximately 1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination.

In trypanosomatid protozoa the biogenesis of mature mRNA involves addition of the spliced leader (SL) sequence from the SL RNA to polycistronic pre-mRNA via trans-splicing. Here we present a mutational analysis of the trypanosomatid Leptomonas collosoma SL RNA to further our understanding of its functional domains important for trans-splicing utilization. Mutant SL RNAs were analyzed for defects in modification of the hypermethylated cap structure (cap 4) characteristic of trypanosomatid SL RNAs, for defects in the first step of the reaction and overall utilization in trans-splicing. Single substitution of the cap 4 nucleotides led to undermethylation of the cap 4 structure, and these mutants were all impaired in their utilization in trans-splicing. Abrogation of the sequence of the Sm-like site and sequences downstream to it also showed cap modification and trans-splicing defects, thus providing further support for a functional linkage between cap modifications and trans-splicing. Further, we report that in L. collosoma both the exon and intron of the SL RNA contribute information for efficient function of the SL RNA in trans-splicing. This study, however, did not provide support for the putative SL RNA-U6 small nuclear RNA (snRNA) interaction at the Sm site like in the nematodes, suggesting differences in the bridging role of U6 in the two trans-splicing systems.

The Saccharomyces cerevisiae Ras proteins control cell growth by regulating the activity of the cAMP-dependent protein kinase (PKA). In this study, a genetic approach was used to identify cellular processes that were regulated by Ras/PKA signaling activity. Interestingly, we found that mutations affecting the C-terminal domain (CTD), of Rpb1p, the largest subunit of RNA polymerase II, were very sensitive to changes in Ras signaling activity. The Rpb1p CTD is a highly conserved, repetitive structure that is a key site of control during the production of a mature mRNA molecule. We found that mutations compromising the CTD were synthetically lethal with alterations that led to elevated levels of Ras/PKA signaling. Altogether, the data suggested that Ras/PKA activity was negatively regulating a protein that functioned in concert with the CTD during RNA pol II transcription. Consistent with this prediction, we found that elevated levels of Ras signaling caused growth and transcription defects that were very similar to those observed in mutants encoding an Rpb1p with a truncated CTD. In all, these data suggested that S. cerevisiae growth control and RNA pol II transcription might be coupled by using the Ras pathway to regulate CTD function.

From transcription to translation, mRNA is complexed with heterogeneous nuclear ribonucleoproteins (hnRNP proteins) that mediate mRNA processing, export from the nucleus, and delivery into the cytoplasm. Although the mechanism is unknown, export of mature mRNA from the nucleus is a critical regulatory step in gene expression. Analyses of hnRNP proteins have shown that many of these proteins are required for this essential cellular process. In this study, we characterize the Saccharomyces cerevisiae Nab2 protein, which was first identified as a poly(A) RNA-binding protein (Anderson, J. T., Wilson, S. M., Datar, K. V., and Swanson, M. S. (1993) Mol. Cell. Biol. 13, 2730-2741). Our work indicates that poly(A) RNA export from the nucleus is dependent upon a functional Nab2 protein; correspondingly, export of Nab2p from the nucleus is dependent upon ongoing RNA polymerase II transcription. Furthermore, we show that Nab2p is modified within its RGG domain by the type I protein-arginine methyltransferase, Hmt1p. Our experiments demonstrate that arginine methylation is required for the export of Nab2p from the nucleus and therefore establish an in vivo effect of this modification. Overall, these experiments provide evidence that Nab2p is an hnRNP protein that is required for poly(A) RNA export and whose export from the nucleus is regulated by Hmt1p.

The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase. Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome. Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities. Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro. The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p. The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage. We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome. We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth. However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.

Interleukin-2 (IL-2) regulates the clonal expansion of activated T cells and is produced in limited amounts during an immune response. Mitogenic induction of human IL-2 gene expression elicits a transient wave of unstable mRNA. We show here that transcription continues unabated during and well beyond the time when the wave is subsiding, yet few, if any, new mRNA molecules are generated once the wave has reached its maximum. Instead, IL-2 precursor transcripts accumulate, becoming the majority of expressed IL-2 RNA molecules. The flow of precursor transcripts into mature mRNA becomes inhibited in the course of induction. When translation is blocked (e.g. by cycloheximide), expression of IL-2 mRNA can be superinduced by 2 orders of magnitude. This superinduction is completely dependent upon transcription, yet is not accompanied by any significant increase in the rate of primary transcription or in mRNA stability. Instead, the processing of nuclear IL-2 precursor transcripts is greatly facilitated, resulting in pronounced superinduction of cytoplasmic mRNA. Once its transcription has been induced, therefore, expression of the IL-2 gene is down-regulated extensively at the level of precursor RNA processing.

A gene encoding the rat kappa opioid receptor (KOR) was cloned and characterized. Results of rat genomic library screening and genomic Southern blot analysis show the gene represented at one copy/haploid genome. Three introns are present within the gene; however, polymerase chain reaction using different sets of primers specifying neighboring exons indicates that alternative splicing does not occur. Using reverse transcription-polymerase chain reaction and primer extension techniques, we are able to demonstrate that two species of transcripts are differentially produced from the KOR gene in a tissue-specific manner. The first transcript that we designate as KOR1 is equivalent to the cDNA sequence reported by other groups and is believed to correspond to KOR subtype 1. KOR1 begins with exon 1 just downstream of two TATA boxes, whereas the second transcript, which we refer to as KORx, begins in intron 1 and thereby retains this intronic sequence in the mature mRNA. Within this intronic sequence there are two potential translation initiation codons that are in-frame with the proposed initiation codon of KOR. The potential open reading frame that starts further upstream in KORx may lead to the translation of a variant KOR protein having a novel peptide sequence at its amino terminus.

Retinoic acid (RA), we show, induces in peripheral blood mononuclear cells a transient wave of newly transcribed, unstable interleukin-1 alpha (IL-1 alpha) and IL-1 beta mRNA. Tumor necrosis factor-alpha mRNA, by contrast, is expressed in multiple waves. IL-1 genes are primary targets for RA. Most IL-1 beta gene transcription induced by RA fails to yield mature mRNA. Instead, precursor transcripts accumulate, detected by ribonuclease protection analysis. The flow of precursors into IL-1 beta mRNA becomes inhibited during induction. When translation is blocked, e.g. by cycloheximide, expression of IL-1 beta mRNA is superinduced by 2 orders of magnitude. Superinduction is dependent on transcription, yet is unaccompanied by increased primary transcription or mRNA stability. Instead, processing of unstable IL-1 beta precursor transcripts into mature mRNA is greatly facilitated. Control is not narrowly localized within precursors: splicing of distinct exons and intron excision are enhanced by cycloheximide. Pre-mRNA processing thus is a limiting step in RA-induced IL-1 beta gene expression. This regulation is specific for RA: when induced by phorbol ester, IL-1 beta gene expression is also superinduced by cycloheximide but that response is accompanied by enhanced mRNA stability. Thus, IL-1 beta gene transcription is induced by RA, yet, unlike for other primary target genes, mRNA expression is regulated at pre-mRNA processing.

The precursor of gonadotropin-releasing hormone (GnRH) and the 56-amino acid GnRH-associated peptide is encoded in an mRNA of about 560 bases in length. This mRNA derives from an approximately 4300-base pair-long gene consisting of four relatively short exons (denoted 1, 2, 3, and 4) and three large introns (A, B, and C). In this study, we characterized the order by which the three introns are spliced from the primary transcript and processing intermediates to give rise to a mature mRNA and evaluated the potential role of gene transcription and pre-mRNA processing in the control of proGnRH mRNA levels in vivo. Nuclear and cytoplasmic RNA fractions isolated from rat preoptic area-anterior hypothalamus (POA-AH) and basal olfactory area (located rostral to the POA) were analyzed by 1) solution hybridization-RNase protection mapping using several RNA probes directed at various regions of the proGnRH gene and 2) reverse transcription-polymerase chain reaction using several oligonucleotide primers. Both types of analysis showed that proGnRH pre-mRNA processing begins with the splicing of intron B from the primary gene transcript. Hence, intron B is the ideal target for studying proGnRH primary transcript by in situ hybridization. Subsequent splicing of introns A and C appeared to take place in two alternative, although not equally prevalent pathways. Quantitative analysis indicated that the proGnRH hnRNA species constituted, on a mole basis, about 20% of the total gene transcripts in the POA-AH. The primary transcript alone constituted about 10% of the total gene transcripts in the POA-AH and as much as 20% in the basal olfactory area. The prospect of blockade of proGnRH hnRNA processing by means of hybridization with endogenous antisense RNAs (transcribed from the SH gene on the opposite strand of the same DNA locus) did not prove to be likely, as the SH transcripts were present at very low levels compared to any of the proGnRH RNA species. We conclude that the relatively large pool of proGnRH hnRNA may reflect a high rate of gene transcription and/or slow RNA processing.

Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145-1157), we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability.

In the rat pituitary, several species of preprolactin (pPrl) mRNA larger than the mature mRNA have been reported (Maurer, R. A., Gubbins, E. J., Erwin, C. R., and Donelson, J. E. (1980) J Biol. Chem. 255, 2243-2246). We examined the size distribution of pPrl transcripts in nuclear, cytoplasmic, and nuclear poly(A)-containing RNA from rat pituitary tumor MtTW10 and from the pituitaries of diethylstilbestrol-treated Fischer 344 X Holtzman rats. RNA was electrophoresed in agarose/2.2 M formaldehyde horizontal slab gels, transferred to diazobenzyloxymethyl paper or to nitro-cellulose paper, and hybridized with a nick-translated recombinant DNA probe containing rat pPrl cDNA sequences. Up to 8 putative precursor mRNA molecules were detected which were from 2 to 13 times the length of mature pPrl mRNA. Three of these RNA species were larger than the maximum size of putative precursors previously reported. These probable precursors were nuclear in origin and polyadenylated, at least in the case of pituitary tumor cells. The largest molecule, 14 kilobase pairs long, may approximate the size of the initial transcript of the prolactin gene.

The synthesis of uteroglobin in the rabbit uterus is induced by progesterone and is repressed by estrogen which has an over-riding effect over the inducer. The dual hormonal control system offers an excellent model for studying hormonal regulation of mammalian gene expression. Using a full-length uteroglobin cDNA clone as a specific hybridization probe, recombinant lambda phages containing the entire chromosomal uteroglobin gene have been isolated from a rabbit genomic DNA library. Electronmicroscopic analysis of hybrid molecules formed between the chromosomal uteroglobin gene and uteroglobin mRNA indicated the presence of 2 intervening sequences within this gene. The mosaic structure of the uteroglobin gene has been substantiated by detailed restriction mapping and Southern hybridization. The gene is 3.0 kilobases in length to code for a mature mRNA of 465 nucleotides. Northern hybridization of poly(A)-containing RNA from 4-day-pregnant rabbit uterus with the full-length cDNA clone revealed the presence of uteroglobin mRNA precursors. The size of the largest precursor RNA species detected by the cDNA clone is the same as the entire chromosomal uteroglobin gene. The fidelity of the precursor RNAs was established by their ability to hybridize with specific intervening sequence probes. Thus the entire uteroglobin gene is expressed into primary RNA transcripts, which are subsequently processed into mature mRNA molecules by splicing.

A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the α1(I) mRNA. The hybrid is digested with RNase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the α1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.

Purified human globin messenger RNA CmRNA) was labeled in vitro with 9 and subjected to digestion with T, and pancreatic RNase. The digests were fractionated in two dimensions and gave characteristic fingerprint patterns. The fingerprints were similar to those obtained with =Plabeled cRNA transcribed from human globin cDNA by RNA polymerase. However, a small number of additional or new oligonucleotides were observed in the ‘7 fingerprints which were either absent or very faint in the fingerprints of the cRNA. Because the lz51 exclusively labels cytidine residues

We have compared the rates of alpha- and beta-globin gene transcription with the rates of mature globin mRNA appearance in the cytoplasm during the course of chemically induced differentiation of mouse erythroleukemia cells by in vivo pulse-labeling experiments. The absolute rates for both processes were determined by simultaneously measuring incorporation into globin-specific transcripts and into the cellular nucleotide pool. The latter measurements provide a determination of the absolute rate of total RNA synthesis which declines during differentiation. Transcription from the beta major and beta minor globin genes was measured separately by hybridization to cloned DNA sequences from a region of the second intron which is highly divergent in the two genes. The results show that, during dimethyl sulfoxide-stimulated differentiation, transcription of alpha and beta major globin increases 15-25-fold, whereas beta minor globin transcription is not increased. Furthermore, in both undifferentiated and differentiated cells, the absolute rates of globin transcription are about equal to the rate of appearance of mature mRNA transcripts in the cytoplasm, indicating highly efficient processing of nuclear globin transcripts to mature mRNA before and after differentiation. The results indicate that dimethyl sulfoxide-induced accumulation of globin mRNA during differentiation is controlled almost entirely at the transcriptional level.

The generation of two discrete mRNAs encoding different polypeptide products in expression of the rat calcitonin, CGRP gene provides an example of developmentally determined alternative RNA processing events in the neuroendocrine system. Both mature mRNA transcripts and their peptide products, encoding either calcitonin or calcitonin gene-related peptide (CGRP) are co-produced in identical thyroid C-cells, in a ratio of 95:1, respectively. These data suggest that the generation of the two mRNAs reflect the actions of a factor(s) dictating alternative RNA processing events.

The mechanism of expression of the structural gene (td) of T4 phage thymidylate synthase, which contains a 1,017-base pair intron, was studied by employing a coupled transcription-translation system with a td containing recombinant plasmid (pKTd2) as template. The [3H]leucine-labeled protein products synthesized in this system were treated with antibody to the synthase and the resulting immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two labeled polypeptides were obtained, one with an Mr of 32,000 and the other with an Mr of 25,000. The former corresponds in molecular weight to a subunit of T4-thymidylate synthase and the other to the 183-amino acid peptide encoded by exon I, the 5'-end of the interrupted td gene. When pKTd2 restricted in exon I was used as a template, labeled immunopeptides were not detected but, when restricted in the intron region or in exon II, only the 25,000 Mr exon I product was obtained. Both peptides (Mr = 25,000 and 32,000) were synthesized when the gene was restricted downstream to exon II. Active enzyme, as measured by the tritium release assay, was shown to form about 6 min after the td gene was added to the in vitro protein synthesizing system, and followed the appearance of mature mRNA, as evidenced by S1 nuclease protection studies. The enzyme increased linearly for another 14 min in conjunction with the appearance of the Mr = 32,000 immunopeptide. The exon I product, however, preceded the Mr = 32,000 peptide, indicating that a post-transcriptional processing event may be required for mature mRNA to be formed. Measurement of the RNA products from the td gene in a transcriptional system, with labeled probes from specific regions of the td gene, provided evidence in support of an RNA processing mechanism involving intron excision and exon splicing.

A messenger RNA preparation from Clostridium pasteurianum is active in stimulating protein synthesis in vitro by ribosomes and initiation factors derived from C. pasteurianum or from Escherichia coli. Other messengers tested, f2 RNA, T4 early mRNA, and E. coli mRNA, are active with ribosomes and initiation factors derived from E. coli, but not with those derived from C. pasteurianum. By testing the heterologous combinations of salt-washed ribosomes and initiation factors from these two bacterial species in protein synthesis assays, it was demonstrated that the salt-washed ribosomes, and not the initiation factors, control the specificity of mRNA translation and also determine the characteristic magnesium optimum for protein synthesis. The initiation factors influence the efficiency of translation obtained with each type of mRNA. The relative efficiency of translation observed when using E. coli initiation factors and when using C. pasteurianum initiation factors varies with the different types of mRNA. The heterologous combinations of salt-washed ribosomes and initiation factors from the two bacterial species were also tested for their ability to bind fMet-tRNA in response to f2 RNA, T4 early mRNA, and C. pasteurianum mRNA. These assays demonstrated the same difference in messenger specificities between the two ribosomal systems as was found in the protein synthesis assays. Moreover, the results indicated, in agreement with the protein synthesis assays, that the salt-washed ribosomes, and not the initiation factors, control the specificity of mRNA recognition.