Margaret MacMorris

Dense Core Vesicle Dynamics in Caenorhabditis elegans Neurons and the Role of Kinesin UNC‐104

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 μm/s−2.7 μm/s.

IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm.

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. J. Comp. Neurol. 429:127–143, 2001.

Intercistronic region required for polycistronic pre-mRNA processing in Caenorhabditis elegans.

ABSTRACT 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-3polycistronic 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 ∼20-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.

Genes involved in pre-mRNA 3'-end formation and transcription termination revealed by a lin-15 operon Muv suppressor screen

RNA polymerase II (Pol II) transcription termination involves two linked processes: mRNA 3′-end formation and release of Pol II from DNA. Signals for 3′ processing are recognized by a protein complex that includes cleavage polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF). Here we identify suppressors encoding proteins that play roles in processes at the 3′ ends of genes by exploiting a mutation in which the 3′ end of another gene is transposed into the first gene of the Caenorhabditis elegans lin-15 operon. As expected, genes encoding CPSF and CstF were identified in the screen. We also report three suppressors encoding proteins containing a domain that interacts with the C-terminal domain of Pol II (CID). We show that two of the CID proteins are needed for efficient 3′ cleavage and thus may connect transcription termination with RNA cleavage. Furthermore, our results implicate a serine/arginine-rich (SR) protein, SRp20, in events following 3′-end cleavage, leading to termination of transcription.

Regulated expression of a vitellogenin fusion gene in transgenic nematodes

In Caenorhabditis elegans the vitellogenin genes are expressed abundantly in the adult hermaphrodite intestine, but are otherwise silent. In order to begin to understand the mechanisms by which this developmental regulation occurs, we used the transformation procedure developed for C. elegans by A. Fire (EMBO. J., 1986, 5, 2673-2680) to obtain regulated expression of an introduced vitellogenin fusion gene. A plasmid with vit-2 upstream and coding sequences fused to coding and downstream sequences of vit-6 was injected into oocytes and stable transgenic strains were selected. We obtained seven independent strains, in which the plasmid DNA is integrated at a low copy number. All strains synthesize substantial amounts of a novel vitellogenin-like polypeptide of 155 kDa that accumulates in the intestine and pseudocoelom, but is not transported efficiently into oocytes. In two strains examined in detail the fusion gene is expressed with correct sex, tissue, and stage specificity. Thus we have demonstrated that the nematode transgenic system can give proper developmental expression of introduced genes and so can be used to identify DNA regulatory regions.

Functional redundancy of worm spliceosomal proteins U1A and U2B

In Caenorhabditis elegans, the small nuclear ribonucleoprotein (snRNP)-associated proteins U1A and U2B″ are ≈50% identical to each other, and neither bears signature characteristics of mammalian U1A or U2B″ or the single Drosophila homolog, SNF. We show here that the genes that encode these proteins (rnp-2 and rnp-3) are cotranscribed in an operon, and that ribonucleoprotein RNP-2 is U1 snRNP-associated (U1A) whereas RNP-3 is U2 snRNP-associated (U2B″). U2B″ interacts with U2 even in the absence of another U2 snRNP protein, U2A′. Like U1A and U2B″ from yeast, plants, and vertebrates, worm U1A and U2B″ are more similar to each other than they are to other U1A or U2B″ proteins, respectively. Even though U1A and U2B″ interact with different snRNPs, they are functionally redundant; knockout of both is required for a lethal phenotype. Interestingly, U1A associates with U2 RNA when U2B″ is deleted. Thus, the two members of this gene family normally function as components of different snRNPs but apparently remain capable of performing the function of the other. Redundancy results from the fact that one protein can substitute for the other, even though it normally does not.