Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m7G-capped RNA is added during mRNA maturation. This short 39 nt m7G-capped RNA, the spliced leader (SL) sequence, is expressed as an ∼100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5′ end of mRNAs by a m7G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as Euglena and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

A single alteration 20 nt 5′ to an editing target inhibits chloroplast RNA editing in vivo

Transcripts of typical dicot plant plastid genes undergo C→U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence –20 to +6 surrounding the C target of editing. When the –20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorporated, virtually no editing of the transcripts occurred in transgenic tobacco plastids. Nucleotides that differ between the black pine and tobacco sequence were tested for their role in C→U editing by designing chimeric genes containing one or more of these divergent nucleotides. Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of the minigene transcript was located –20 nt 5′ to the C target of editing. Expression of transgene transcripts carrying the 27 nt sequence did not affect the editing extent of the endogenous rpoB transcripts, even though the chimeric transcripts were much more abundant than those of the endogenous gene. In plants carrying a 93 nt rpoB editing site sequence, transgene transcripts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of the 27 nt site for a trans-acting factor and lower abundance of the transcript could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing.

Identification of the yeast cytidine deaminase CDD1 as an orphan C→U RNA editase

Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion. The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast. Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs. Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C→U editing on a reporter mRNA. This is only the second report of a cytidine deaminase that can use mRNA as a substrate. CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity. Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1. Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected. We propose that CDD1 be designated an orphan C→U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes.

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.

Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.

Evolution of RNA editing in trypanosome mitochondria

Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs encoded in both the maxicircle and the minicircle molecules and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C34 to U34 modification in the anticodon of the imported tRNATrp, thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and appears to have evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for the evolutionary fixation of these retroposition events was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded guide RNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in the relative abundance of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The possible evolutionary relationship of the C to U and U-insertion editing systems is discussed.

Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans

Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm–oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in C. elegans.

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

The intercistronic region between the maturation and coat-protein genes of RNA phage MS2 contains important regulatory and structural information. The sequence participates in two adjacent stem-loop structures, one of which, the coat-initiator hairpin, controls coat-gene translation and is thus under strong selection pressure. We have removed 19 out of the 23 nucleotides constituting the intercistronic region, thereby destroying the capacity of the phage to build the two hairpins. The deletion lowered coat-protein yield more than 1000-fold, and the titer of the infectious clone carrying the deletion dropped 10 orders of magnitude as compared with the wild type. Two types of revertants were recovered. One had, in two steps, recruited 18 new nucleotides that served to rebuild the two hairpins and the lost Shine-Dalgarno sequence. The other type had deleted an additional six nucleotides, which allowed the reconstruction of the Shine-Dalgarno sequence and the initiator hairpin, albeit by sacrificing the remnants of the other stem-loop. The results visualize the immense genetic repertoire created by, what appears as, random RNA recombination. It would seem that in this genetic ensemble every possible new RNA combination is represented.

Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.

Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants. In this study, microinjection protocols were used to directly examine cell-to-cell trafficking of CMV viral RNA in these resistant plants. CMV RNA fluorescently labeled with the nucleotide-specific TOTO-1 iodide dye, when coinjected with unlabeled CMV 3a movement protein (MP), moved rapidly into the surrounding mesophyll cells in mature tobacco leaves of vector control and untransformed plants. Such trafficking required the presence of functional CMV 3a MP. In contrast, coinjection of CMV 3a MP and CMV TOTO-RNA failed to move in transgenic resistant plants expressing the CMV truncated replicase gene. Furthermore, coinjection of 9.4-kDa fluorescein-conjugated dextran (F-dextran) along with unlabeled CMV 3a MP resulted in cell-to-cell movement of the F-dextran in control plants, but not in the transgenic plants. Similar results were obtained with viral RNA when the 30-kDa MP of tobacco mosaic virus (TMV) was coinjected with TMV TOTO-RNA into replicase-resistant transgenic tobacco expressing the 54-kDa gene sequence of TMV. However, in these transgenic plants, the TMV-MP was still capable of mediating cell-to-cell movement of itself and the 9.4-kDa F-dextran. These results indicate that an inhibition of cell-to-cell viral RNA trafficking is correlated with replicase-mediated resistance. This raises the possibility that the RNA-2 product is potentially involved in the regulation of cell-to-cell movement of viral infectious material during CMV replication.

Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus.

A newly recognized gamma herpesvirus known as Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is present in Kaposi sarcomas and body-cavity-based lymphomas. Here we identify a novel abundant 1.2-kb RNA, polyadenylated nuclear RNA (PAN RNA), encoded by the virus. The majority of cDNAs produced from poly(A)-selected RNA isolated from a human body cavity lymphoma cell line 48 hr after butyrate induction of KSHV lytic replication represented PAN RNA. Within PAN RNA were two 9 and 16 nt stretches with 89% and 94% identity to U1 RNA. A third stretch of 14 nt was 93% complementary to U1. The 5' upstream region of PAN RNA contained both proximal and distal sequence elements characteristic of regulatory regions of U snRNAs, whereas the 3' end was polyadenylylated. PAN RNA was transcribed by RNA polymerase II, lacked a trimethylguanosine cap, and did not associate with polyribosomes. PAN RNA formed a speckled pattern in the nucleus typical of U snRNAs and colocalized with Sm protein. Therefore, PAN represents a new type of RNA, possessing features of both U snRNA and mRNA.

Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.