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.

In vivo function of mutated spliced leader RNAs in Caenorhabditis elegans

The role of spliced leader RNA (SL RNA) in trans-splicing in Caenorhabditis elegans has been studied through a combination of in vitro mutagenesis and in vivo complementation of rrs-1 mutant nematodes, which lack endogenous SL1 RNA. Three classes of mutant SL1 RNAs have been found—those that rescue the lethal phenotype at low concentration of transforming DNA, those that rescue at high but not low concentration, and those that do not rescue at all. These studies showed that some mutations in the otherwise highly conserved 22-nt spliced leader are tolerated for splicing and post-splicing events. A longer spliced leader also can be tolerated but only when present in high copy number. Changes in the first 16 nucleotides result in the appearance of no SL RNA, consistent with the in vitro studies by others showing that the SL1 RNA promoter partly resides within the spliced leader sequence.

Trans-spliced leader addition to mRNAs in a cnidarian

A search of databases with the sequence from the 5′ untranslated region of a Hydra cDNA clone encoding a receptor protein-tyrosine kinase revealed that a number of Hydra cDNAs contain one of two different sequences at their 5′ ends. This finding suggested the possibility that mRNAs in Hydra receive leader sequences by trans-splicing. This hypothesis was confirmed by the finding that the leader sequences are transcribed as parts of small RNAs encoded by genes located in the 5S rRNA clusters of Hydra. The two spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucleotides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Both of the SL RNAs are bound by antibody against trimethylguanosine, suggesting that they contain a trimethylguanosine cap. The predicted secondary structures of the Hydra SL RNAs show significant differences from the structures predicted for the SLs of other organisms. Messenger RNAs have been identified that can receive either SL-A or -B, although the impact of the two different SLs on the function of the mRNA is unknown. The presence and features of SL addition in the phylum Cnidaria raise interesting questions regarding the evolution of this process.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei

In Trypanosoma brucei, transcription by RNA polymerase II and 5′ capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.

A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.

Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon

The deg-3 gene from the nematode Caenorhabditis elegans encodes an α subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an α subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

Alternatively spliced N resistance gene transcripts: Their possible role in tobacco mosaic virus resistance

The N gene, a member of the Toll-IL-1 homology region–nucleotide binding site–leucine-rich repeat region (LRR) class of plant resistance genes, encodes two transcripts, NS and NL, via alternative splicing of the alternative exon present in the intron III. The NS transcript, predicted to encode the full-length N protein containing the Toll-IL-1 homology region, nucleotide binding site, and LRR, is more prevalent before and for 3 hr after tobacco mosaic virus (TMV) infection. The NL transcript, predicted to encode a truncated N protein (Ntr) lacking 13 of the 14 repeats of the LRR, is more prevalent 4–8 hr after TMV infection. Plants harboring a cDNA-NS transgene, capable of encoding an N protein but not an Ntr protein, fail to exhibit complete resistance to TMV. Transgenic plants containing a cDNA-NS-bearing intron III and containing 3′ N-genomic sequences, encoding both NS and NL transcripts, exhibit complete resistance to TMV. These results suggest that both N transcripts and presumably their encoded protein products are necessary to confer complete resistance to TMV.

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

Gene recognition via spliced sequence alignment.

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

Proteolysis of major histocompatibility complex class II-associated invariant chain is regulated by the alternatively spliced gene product, p41.

Invariant chain (Ii) is an intracellular type II transmembrane glycoprotein that is associated with major histocompatibility complex class II molecules during biosynthesis. Ii exists in two alternatively spliced forms, p31 and p41. Both p31 and p41 facilitate folding of class II molecules, promote egress from the endoplasmic reticulum, prevent premature peptide binding, and enhance localization to proteolytic endosomal compartments that are thought to be the sites for Ii degradation, antigen processing, and class II-peptide association. In spite of the dramatic and apparently equivalent effects that p31 and p41 have on class II biosynthesis, the ability of invariant chain to enhance antigen presentation to T cells is mostly restricted to p41. Here we show that degradation of Ii leads to the generation of a 12-kDa amino-terminal fragment that in p41-positive, but not in p31-positive, cells remains associated with class II molecules for an extended time. Interestingly, we find that coexpression of the two isoforms results in a change in the pattern of p31 degradation such that endosomal processing of p31 also leads to extended association of a similar 12-kDa fragment with class II molecules. These data raise the possibility that p41 may have the ability to impart its pattern of proteolytic processing on p31 molecules expressed in the same cells. This would enable a small number of p41 molecules to modify the post-translational transport and/or processing of an entire cohort of class II-Ii complexes in a manner that could account for the unique ability of p41 to enhance antigen presentation.

A third member of the synapsin gene family

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12–13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.