Identification and structural characterization of a cDNA clone encoding a membrane-bound form of the polypeptide pheromone Er-1 in the ciliate protozoan Euplotes raikovi.

Abstract

In the ciliate Euplotes raikovi, the same cell that secretes the pheromone Er-i, a polypeptide of 40 amino acids derived from a precursor (prepro-Er-1) of 75 amino acids, also produces a polypeptide of 130 amino acids, of which the 75 residues at the carboxyl terminus are identical to those of prepro-Er-1 and the 55 residues at the amino terminus form a new sequence. This larger Er-i isoform is retained in membranes, where it may function as a binding site for soluble Er-i in a mechanism of autocrine secretion. Membrane-bound and soluble Er-i are translated from two mRNAs that apparently originate from a common micronuclear and/or macronuclear gene through alternative elimination of intervening sequences. This finding suggests that single genes responsible for the generation of isoform diversity in polypeptide hormones are present even in single-celled eukaryotes.

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

“…This peculiar organization of the E. nobilii pheromone genes probably reflects a conserved inclusion of intron sequences, whose splicing might have a fundamental role in the mechanism of expression of these genes. This hypothesis receives support in particular from previous functional analysis of E. raikovi pheromone genes [26,27], showing that the primary transcripts of these genes undergo intron splicing (at not canonical sites) to generate membranebound pheromone isoforms that cells use as autocrine pheromone receptors [9]. Its assessment, in addition to being crucial to elucidate the mechanism of expression of the E. nobilii pheromone gene family, may also contribute insightful information on the more general problem of the functional organization of the sub-chromosomic, gene-sized macronuclear genomes of the hypotrich ciliates.…”

“…As is the case in pheromone genes of other species of Euplotes [14,26,27], these functions are presumably correlated with the inclusion of intron sequences the splicing of which results in the expression of new ORF's and the synthesis of new and functionally diversified pheromone isoforms. This possibility is supported by the common conservation in the 5' regions of, (i) multiple ATG start codons, (ii) A + T rich sequences, and (iii) consensus donor GT (5') and acceptor AG (3') splice-site junctions [28,29].…”

Developmentally and environmentally regulated expression of gamone 1: the trigger molecule for sexual reproduction inBlepharisma japonicum

“…In the ciliate Euplotes raikovi, mating pheromones (euplomones) are synthesized constitutively throughout the entire clonal life cycle and in growing cells (Miceli et al, 1989;Luporini et al, 1992;Miceli et al, 1992;Vallesi et al, 1995;Luporini et al, 1995;Ortenzi et al, 2000). The gene encoding each pheromone generates at least two distinct molecules by alternative splicing, one of which is the soluble pheromone and the other a membrane-bound pheromone isoform.…”

Autocrine, Mitogenic Pheromone Receptor Loop of the Ciliate Euplotes raikovi : Pheromone-Induced Receptor Internalization

“…The second transcript includes the prepropheromone sequence and a novel N-terminal sequence. When its translation product is processed, the entire C-terminal prepropheromone is retained as an extracellular peptide anchored to the cell surface through the "pre" transmembrane segment, and the N terminus is intracellular (13,20). It is this membrane-bound pheromone isoform that cells utilize as a specific pheromone receptor (24).…”

“…When cells bind a nonself pheromone from another cell type, they temporarily arrest their growth and develop competence for uniting in mating pairs (23). We refer to the response to self pheromones as an autocrine response and the response to nonself pheromones as a paracrine response.The field addressing the issue of how cells can discriminate between autocrine and paracrine pheromone binding and accordingly mount a growth or mating response was advanced by the identification of the pheromone receptors as part of a study of pheromone gene structure and expression (20). These genes, which are transcriptionally active in thousands of copies in the somatic cell macronucleus (15), generate primary transcripts that undergo a process of alternative splicing that creates two closely related transcripts, one of which codes for the prepropheromone.…”