Coupling of Tetrahymena ribosomal RNA splicing to beta-galactosidase expression in Escherichia coli.

Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any protein in vitro. The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid. Production of functional beta-galactosidase is dependent on RNA splicing in vivo in Escherichia coli. Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA. The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of which have been characterized.

Origin of the phosphate at the ligation junction produced by self-splicing of Tetrahymena thermophila pre-ribosomal RNA.

The exons of the self-splicing pre-ribosomal RNA of Tetrahymena thermophila are joined accurately in vitro, even when only 33 nucleotides of the natural 5' exon and 38 nucleotides of the natural 3' exon remain. RNA fingerprint analysis was used to identify the unique ribonuclease T1 oligonucleotide generated by exon ligation. Secondary digests of the ligation junction oligonucleotide with ribonuclease A confirmed the identity of the fragment and demonstrated that the phosphate group that forms the phosphodiester bond at the ligation junction is derived from the 5' position of a uridine nucleotide in the RNA. This observation supports the prediction that the splice junction phosphate is derived from the 3' splice site. These results emphasize the mechanistic similarities of RNA splicing reactions of the group I introns, group II introns and nuclear pre-mRNA introns.

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon.

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.

Dominant enhancers of Egfr in Drosophila melanogaster: genetic links between the Notch and Egfr signaling pathways.

The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F1 screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context.

Expression of protein tyrosine phosphatase genes during oogenesis in Drosophila melanogaster.

The spatial and temporal expression of seven Drosophila protein tyrosine phosphatase genes during oogenesis was examined by whole mount in-situ hybridization of antisense RNA probes to ovaries. Our observations indicate diverse expression patterns consistent with multiple roles for protein tyrosine phosphatases in the ovary. DPTP99A and corkscrew transcripts are expressed in follicle cells, consistent with possible roles in the EGF receptor signaling pathway. Transcripts from corkscrew and DPTP10D are detected in the germline during oogenesis and localized to the oocyte during egg chamber development. Localization of the two transcripts is disrupted by mutations in egalitarian and Bicaudal D. DLAR and DPTP4E transcripts are found in the germline during the same developmental stages as DPTP10D transcripts, but their transcripts are not localized to the oocyte. DPTP61F transcription is detected only after stage 6 of oogenesis. After stage 10B these transcripts are transported to the oocyte; thus ovarian transcription of DPTP61F may reflect a maternal contribution of the mRNA for later use during embryogenesis. DPTP69D transcripts are sequestered in the nucleus from stage 7 to stage 10, and then released to the cytoplasm. Our observations suggest that the export of DPTP69D mRNA from the nucleus is temporally regulated during oogenesis.

Differential accumulation of DPTP61F alternative transcripts: regulation of a protein tyrosine phosphatase by segmentation genes.

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. DPTP61Fn accumulates in the nucleus, and DPTP61Fm associates with the membranes of the reticular network and the mitochondria. We have examined the spatial and temporal expression of the two alternative transcripts of dptp61F during Drosophila embryogenesis. Our observations indicate that the two isoforms are expressed in distinct patterns. The DPTP61Fn transcript is expressed in the mesoderm and neuroblast layer during germband extension and later in the gut epithelia. In comparison, the transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression in the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation in the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes.

Determinants of the 3' splice site for self-splicing of the Tetrahymena pre-rRNA.

Tetrahymena preribosomal RNA undergoes self-splicing in vitro. The structural components involved in recognition of the 5' splice site have been identified, but the mechanism by which the 3' splice site is recognized is not established. To identify some components of 3'splice site recognition, we have generated mutations near the 3' splice site and determined their effects on self-splicing. Alteration of the 3'-terminal guanosine of the intervening sequence (IVS), a conserved nucleotide in group I IVSs, almost eliminated 3' splice site activity; the IVS-3' exon splicing intermediate accumulated, and exon ligation was extremely slow. These mutations do not result in recruitment of cryptic 3' splice sites, in contrast to mutations that affect the 5' splice site. Alteration of the cytidine preceding the 3'-terminal guanosine or of the first two nucleotides of the 3' exon had similar but less severe effects on exon ligation. Most of the mutants showed some reduction (less than threefold) in GTP addition at the 5' splice site. A mutation that placed a new guanosine residue just upstream from the 3'-terminal guanosine misspliced to produce ligated exons with one extra nucleotide between the 5' and 3' exons. We conclude that multiple nucleotides, located both at the 3' end of the IVS and in the 3' exon, are required for 3' splice site recognition.

The maternal ventralizing locus torpedo is allelic to faint little ball, an embryonic lethal, and encodes the Drosophila EGF receptor homolog.

The torpedo gene of Drosophila melanogaster is involved in the establishment of the dorsoventral pattern of eggshell and embryo. We have isolated new alleles of torpedo and have found that torpedo is allelic to the zygotic embryonic lethal faint little ball. We have shown that torpedo resides in subdivision 57F on the second chromosome--at the same location as the Drosophila homolog of the EGF receptor (DER). Using a cosmid that contains most of the DER coding region as a hybridization probe, we have shown that a cytologically small deficiency that eliminates torpedo activity also removes the DER gene, and that an inversion that was isolated as a strong torpedo allele breaks the coding region of the DER gene. We conclude that torpedo is the DER gene.

A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA.

The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site. Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing.