We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes. The post-translational processing of IDA-1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL-3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time-lapse epifluorescence microscopy of IDA-1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC-104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 mm/sÀ2.7 mm/s.
Expression of a gfp transgene in the intestines of living Caenorhabditis elegans has been measured following depletion by RNAi of a variety of known splicing factors and mRNA export proteins. Reduction of most splicing factors showed only a small effect on expression of the transgene in the animal injected with dsRNA, although most of these RNAi's resulted in embryonic lethality in their offspring. In contrast, RNAi of nxf-1, the worm homolog of mammalian NXF1/TAP, a key component of the mRNA export machinery, resulted in dramatic suppression of GFP expression in the injected animals. When we tested other proteins previously reported to be involved in marking mRNAs for export, we obtained widely divergent results. Whereas RNAi of the worm REF/Aly homologs had no obvious effect, either in the injected animals or their offspring, RNAi of UAP56, reported to be the partner of REF/Aly, resulted in strong suppression of GFP expression due to nuclear retention of its mRNA. Overexpression of UAP56 also resulted in rapid loss of GFP expression and lethality at all stages of development. We conclude that UAP56 plays a key role in mRNA export in C. elegans, but that REF/Aly may not. It also appears that some RNA processing factors are required for viability (e.g., U2AF, PUF60, SRp54, SAP49, PRP8, U1-70K), whereas others are not (e.g., U2A, CstF50).
The closely related mammalian proteins IA-2 and phogrin are protein tyrosine phosphatase-like receptor proteins spanning the membrane of dense core vesicles of neuroendocrine tissues. They are of interest as molecular components of the secretory machinery and as major targets of autoimmunity in type I diabetes mellitus. The Caenorhabditis elegans genome has a single copy of an IA-2/phogrin homolog ida-1 III (islet cell diabetic autoantigen), which encodes the ida-1 (B0244.2) gene product as a series of 12 exons over a 10-kb region of chromosome III. The full-length sequence of the ida-1 cDNA encoded a 767-amino acid type 1 transmembrane protein of 87 kDa. The PTP catalytic site consensus sequence of IDA-1, like IA-2 and phogrin, diverged and would not be active. Expression of green fluorescent protein (GFP) under the ida-1 gene promoter showed activity in a subset of around 30 neurons with sensory functions and the uv1 cells of the vulva in hermaphrodites. Males showed additional expression in male-specific neurons. In situ experiments in rat brain showing the distribution of IA-2 and phogrin suggested a complimentary and overlapping pattern compared with the proprotein convertases PC1 and PC2. In C. elegans, IDA-1-expressing cells comprised a subset of those expressing the PC2 homolog KPC-2 (C51E3. 7), consistent with IDA-1 being a component of neuropeptide-containing dense core vesicles. The results support the hypothesis that C. elegans IDA-1 is the functional homolog of IA-2 and phogrin in mammals. Analysis of the function of IDA-1 should contribute to our understanding of the function of these proteins in signal transduction, vesicle locomotion, and exocytosis.
In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3 ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5 ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2-gpd-3 polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3 end were important for 3-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3-end formation was shown to be important for downstream mRNA biosynthesis. This ϳ20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.Two characteristic features of Caenorhabditis elegans make it a unique model system among eukaryotes for studying RNA processing. First, approximately 70% of the genes in C. elegans undergo trans splicing during processing of the pre-mRNA. trans splicing involves the transfer of a 22-nucleotide (nt) spliced leader (SL) sequence from the SL snRNP to the 5Ј ends of the mRNAs (2). The majority of trans splicing utilizes SL1 RNA and most SL1 trans splicing occurs near the 5Ј ends of pre-mRNAs that begin with an outron, an AU-rich intronlike sequence containing a functional 3Ј splice site (UUUUC AG/R) but lacking a 5Ј splice site (5-7). Second, many C. elegans genes are arranged in operons (16,21). These genes are found in closely linked gene clusters that are cotranscribed to produce polycistronic pre-mRNAs. Processing of these polycistronic precursors into mature monocistronic transcripts involves a combination of cleavage and polyadenylation at the 3Ј end of the upstream mRNA and trans splicing at the 5Ј end of the downstream mRNA. A second type of SL snRNP, called SL2, is used exclusively for trans splicing to the downstream genes in these polycistronic transcripts (16, 21), although mRNAs from some downstream genes in operons are trans spliced to both SL1 and SL2 (2). Since the discovery of operons and SL2 trans splicing in C. elegans, they have been found in other nematodes, including Caenorhabditis briggsae (13) and Dolichorhabditis (9). We do not know how widespread operons are in eukaryotes, although polycistronic transcripts have been identified in a variety of organisms, including Drosophila melanogaster and mammals (1).Although the general splicing machinery is conserved in C. elegans (2), the existence of operons and trans splicing suggests there could be some machinery specific for them. Part of the trans splicing machinery, the SL snRNP, has been analyzed in Ascaris, another nematode (8). However, we know little about the unique mac...
UAP56 is an essential eukaryotic pre-mRNA splicing factor and mRNA export factor. The mechanisms of its functions are not well understood. We determined the crystal structures of the N- and C-terminal domains of human UAP56 (comprising 90% of the full-length UAP56) at 1.9 A resolution. The two domains each have a RecA-like fold and are connected by a flexible linker. The overall fold of each domain is highly similar to the corresponding domains of eIF4A (a prototypic DExD/H-box protein), with differences at the loops and termini. This structural similarity suggests that UAP56 is likely to possess ATPase and helicase activity similar to eIF4A. The NTP binding pocket of UAP56 is occupied by a citrate ion, mimicking the phosphates of NTP and retaining the P loop in an open conformation. The crystal structure of the N-terminal domain of UAP56 also reveals a dimer interface that is potentially important for UAP56's function.
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