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Complex biological processes require coordinated function of many genes. One evolutionary solution to the problem of coordinately expressing functionally related genes in bacteria and nematodes is organization of genes in operons. Surprisingly, eukaryotic operons are considered rare outside the nematode lineage. In Drosophila melanogaster , we found lounge lizard ( llz ), which encodes a degenerin/ENaC cation channel, cotranscribed with CheB42a , a nonhomologous gene of unknown function residing Drosophila operons. These data reveal eukaryotic operon-like transcription of functionally related genes in Drosophila . The results also suggest that operon-based transcription may be more common in eukaryotes than previously appreciated.

The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate–early gene Arc . Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at ≈40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

MicroRNAs (miRNAs) regulate posttranscriptional gene activity by binding to specific sequences in the 3′ UTRs of target mRNAs. A number of metazoan miRNAs have been shown to exhibit tissue-specific patterns of expression. Here, we investigate the possibility that localized expression is mediated by tissue-specific enhancers, comparable to those seen for protein-coding genes. Two miRNA loci in Drosophila melanogaster are investigated, the mir-309–6 polycistron (8-miR) and the mir-1 gene. The 8-miR locus contains a cluster of eight distinct miRNAs that are transcribed in a common precursor RNA. The 8-miR primary transcript displays a dynamic pattern of expression in early embryos, including repression at the anterior and posterior poles. An 800-bp 5′ enhancer was identified that recapitulates this complex pattern when attached to a RNA polymerase II core promoter fused to a lacZ -reporter gene. The miR-1 locus is specifically expressed in the mesoderm of gastrulating embryos. Bioinformatics methods were used to identify a mesoderm-specific enhancer located ≈5 kb 5′ of the miR-1 transcription unit. Evidence is presented that the 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist. These results provide evidence that restricted activities of the 8-miR and miR-1 miRNAs are mediated by classical tissue-specific enhancers.

Fn containing an extra type III domain (EIIIA in the rat, ED1 or EDA in humans) is commonly termed “fetal” fibronectin, but it is prominent during the injury response of adult tissues and mediates important early events in the response. This form is particularly apparent in acute liver injury, where it has been shown that sinusoidal endothelial cells produce EIIIA-fibronectin. This fibronectin isoform arises by alternative splicing of the primary transcript. In the present experiments, we have studied the regulation of fibronectin splicing in primary sinusoidal endothelial cells by transfecting a minigene containing the EIIIA exon and its flanking introns, driven by various promoters. The results indicate that fibronectin splicing in endothelial cells from normal liver is in part promoter-dependent. However, in cells from injured liver in which expression of both total and EIIIA-fibronectin is strikingly increased, promoter effects disappear. Because fibronectin splicing is known to be regulated in part by TGFβ, we also examined the effect of a soluble inhibitor of the TGFβ type 2 receptor. This agent had no effect on splicing by normal endothelial cells. By contrast, for endothelial cells from the injured liver, the splicing pattern reverted to that of normal cells, i.e., it became promoter-dependent. We conclude that, in the setting of injury in vivo , TGFβ overrides the promoter dependence of fibronectin splicing in normal cells. The data suggest that TGFβ modifies the spliceosome, if not through its known signaling intermediates, then through the products of genes regulated by this cytokine.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

Thus far, only one major form of vertebrate DNA (cytosine-5) methyltransferase (CpG MTase, EC 2.1.1.37 ) has been identified, cloned, and extensively studied. This enzyme, dnmt1, has been hypothesized to be responsible for most of the maintenance as well as the de novo methylation activities occurring in the somatic cells of vertebrates. We now report the discovery of another abundant species of CpG MTase in various types of human cell lines and somatic tissues. Interestingly, the mRNA encoding this CpG MTase results from alternative splicing of the primary transcript from the Dnmt1 gene, which incorporates in-frame an additional 48 nt between exons 4 and 5. Furthermore, this 48-nt exon sequence is derived from the first, or the most upstream, copy of a set of seven different Alu repeats located in intron 4. The ratios of expression of this mRNA to the expression of the previously known, shorter Dnmt1 mRNA species, as estimated by semiquantitative reverse transcription–PCR analysis, range from two-thirds to three-sevenths. This alternative splicing scheme of the Dnmt1 transcript seems to be conserved in the higher primates. We suggest that the originally described and the recently discovered forms of CpG MTase be named dnmt1-a and dnmt1-b, respectively. The evolutionary and biological implications of this finding are discussed in relation to the cellular functions of the CpG residues and the CpG MTases.

The two putative ribonucleotide reductase subunits of the Bacillus subtilis bacteriophage SPβ are encoded by the bnrdE and bnrdF genes that are highly similar to corresponding host paralogs, located on the opposite replication arm. In contrast to their bacterial counterparts, bnrdE and bnrdF each are interrupted by a group I intron, efficiently removed in vivo by mRNA processing. The bnrdF intron contains an ORF encoding a polypeptide similar to homing endonucleases responsible for intron mobility, whereas the bnrdE intron has no obvious trace of coding sequence. The downstream bnrdE exon harbors an intervening sequence not excised at the level of the primary transcript, which encodes an in-frame polypeptide displaying all the features of an intein. Presently, this is the only intein identified in bacteriophages. In addition, bnrdE provides an example of a group I intron and an intein coding sequence within the same gene.

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

Sex-specific alternative processing of double-sex (dsx) precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila melanogaster. By transfection analyses using dsx minigene constructs, we identified six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA in the female-specific fourth exon that act as the cis elements for the female-specific splicing of dsx pre-mRNA. UV-crosslinking experiments revealed that both female-specific transformer (tra) and transformer-2 (tra-2) products bind to the 13-nucleotide sequences of dsx pre-mRNA. These results strongly suggest that the female-specific splicing of dsx pre-mRNA is activated by binding of these proteins to the 13-nucleotide sequences.

A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an approximately 110-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a greater than 150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs--the SL-RNA and U-RNAs--consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping.

The maturation and quality control of mRNA in eukaryotes is a tightly regulated, multistep process that begins on nascent transcripts. A 7-methyl guanosine (7MeG) cap structure is added to the 5′ end of pre-mRNA as it emerges from the exit channel of RNA Polymerase II. The multifunctional nuclear cap-binding complex (CBC), consisting of two protein subunits (CBP80 and CBP20), assembles at the pre-mRNA cap early during transcript formation and helps recruit the splicesome machinery to the cap-proximal intron (1, 2). Termination of transcription involves cleavage and polyadenylation at the 3′ end, and the mature mRNA is retained in the nucleus or exported to the cytoplasm. In either case, the mRNA undergoes a pioneer round of translation and surveillance by the nonsense-mediated mRNA decay (NMD) pathway to eliminate defective or misspliced transcripts. Although the CBC localizes primarily to the nucleus, it remains associated with mRNAs during export to the cytoplasm and during the pioneer round of translation and mRNA surveillance. After the first round of translation, the CBC is replaced by the eukaryotic initiation complex eIF4F, and the mRNA steady-state translation initiation complex is formed (3). But not all RNA Pol II transcripts are predestined for translation. Primary transcripts for microRNA (pri-miRNA) are retained in the nucleus, where they are processed into ≈21- to 22-nt miRNA that generally function as posttranscriptional regulators of mRNA expression. Although it is known that pri-miRNA transcripts form by RNA Pol II transcription, the extent to which pri-miRNA and pre-mRNA transcripts share common processing components is far from settled. In this issue of PNAS, Laubinger et al. (4) identify an important relationship between mRNA maturation and miRNA primary transcript processing in plants.

In most eukaryotic cells, the C-terminal amino acid of α-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of α-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin–Tyr ligase. There is evidence that tubulin–Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

UV transcription mapping with recombinant DNA probes containing immunoglobulin kappa light chain mRNA sequences has been used to determine the size of the transcription unit coding for kappa light chain m RNA and to establish the arrangement of variable and constant regions in this transcription unit. In relation to ribosomal RNA standards, the transcription of kappa light chain constant region sequences into nuclear RNA exhibits a UV target size of 9.6 kbases (kb). The kappa light chain variable region exhibits a UV target size of 7.6 kb indicating that it is separated by approximately 2.0 kb from the constant region in the kappa light chain transcription unit. The size of the primary transcript (i.e., the direct, unprocessed RNA product of transcription) predicted from the constant region target size concurs with our previous pulse-labeling results which showed that the largest presumptive nuclear RNA precursor to kappa light chain mRNA is approximately 10 kb. In addition, the UV target size of cytoplasmic kappa mRNA is indistinguishable from the target size of constant region sequences in nuclear RNA. These results suggest that the kappa light chain transcription unit is copied directly into a 10-kb nuclear RNA precursor in which the kappa variable and constant regions are separated by approximately 2 kb. Accordingly, it is proposed that the joining of immunoglobulin kappa light chain variable and constant regions occurs in the post-transcriptional processing of this large nuclear RNA precursor into kappa light chain mRNA.

All sequenced histidine tRNAs have one additional nucleotide at the 5' end when compared to other tRNA species. Sequence analysis of histidine tRNA genes from Drosophila melanogaster and Schizosaccharomyces pombe showed that the terminal guanylate residue of the mature tRNAs is not encoded by the genes. Analysis of the products from in vitro transcription of these genes in extracts from Drosophila Kc cells demonstrated that the 5'-terminal nucleotide present in the mature tRNA is added post-transcriptionally. The addition reaction requires ATP. A portion of the mature tRNAs are then modified at the 5'-terminal pG. Analysis of the RNA species formed during the in vitro maturation of the Drosophila histidine tRNA primary transcript uncovered the following maturation scheme: (i) the primary transcript is processed by RNase P at the 5' end to form an intermediate precursor; (ii) the 3'-flanking sequence is endonucleolytically removed, and a guanylate moiety is added to the 5' end to form mature-sized histidine tRNA; and (iii) a fraction of the 5'-terminal guanylate residues then undergoes modification. In contrast to the capping of eukaryotic mRNA, the guanylate addition to histidine tRNA results in the formation of a (3'-5')-phosphodiester bond. There are no precedents for the post-transcriptional addition of nucleotides (in phosphodiester linkage) to the 5' end of RNA precursors.

RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript. In mutants deficient in RNase III, no species cleaved by RNase III are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely. Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA. Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth. Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase III were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced.

We report the results of a novel method for locating sites of transcription initiation using a complex of capping enzymes from vaccinia virions that catalyze the reaction pppG + S -adenosylmethionine + (p)ppXpYpZp..... → 7m GpppXpYpZp..... [Ensinger, M. J., Martin, S. A., Paoletti, E. and Moss, B. (1975) Proc. Natl. Acad. Sci. USA 72, 2525-2529]. This enzyme complex will cap di- or triphosphate termini but will not cap monophosphate or hydroxyl termini. Xenopus laevis 40S precursor rRNA from oocytes is capped by these enzymes, and we conclude that it has 5′-polyphosphate termini. Therefore, 40S RNA must represent the primary transcript of amplified X. laevis ribosomal DNA. The majority of 40S molecules with polyphosphate termini begin with the sequence (p)ppAAG. There is evidence, however, that the 5′ terminus may be heterogeneous. The majority of all detectable initiation events were localized close to the region coding for the 5′ end of the 40S RNA. No initiation sites were detected in the nontranscribed spacer, but an apparent initiation site in the middle of the transcribed region was also observed.