Mechanism of GnRH receptor signaling on gonadotropin release and gene expression in pituitary gonadotrophs.

Gonadotropin releasing hormone (GnRH), the first key hormone of reproduction, is synthesized and secreted from the hypothalamus in a pulsatile manner and stimulates pituitary gonadotrophs (5-10% of the pituitary cells) to synthesize and release gonadotropin luteinizing hormone (LH) and follicle stimulating hormone (FSH). Gonadotrophs consist of 60% multihormonal cells (LH+FSH) and 18% LH- and 22% FSH-containing cells. LH and FSH, members of the glycoprotein hormone family, stimulate spermatogenesis, folliculogenesis, and ovulation. Although GnRH plays a pivotal role in gonadotropin synthesis and release, other factors such as gonadal steroids and gonadal peptides exert positive and negative feedback mechanisms, which affect GnRH actions. GnRH actions include activation of phosphoinositide turnover as well as phospholipase D and A2, mobilization and influx of Ca2+, activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). A complex crosstalk between the above messenger molecules mediates the diverse actions of GnRH. Understanding the signaling mechanisms involved in GnRH actions is the basis for our understanding of basic reproductive functions in general and gonadotropin synthesis and release in particular.

RNA editing associated with the generation of two distinct conformations of the trypanosomatid Leptomonas collosoma 7SL RNA.

Analysis of the trypanosomatid Leptomonas collosoma 7SL RNA revealed the existence of two distinct stable 7SL RNA conformers (7SL I and II). Sequence analysis of the RNAs indicated a single base difference between the conformers at position 133 (C in 7SL II and U in 7SL I) located in domain III. This change appears to be the result of a post-transcriptional editing event, since the single-copy 7SL RNA gene codes exclusively for a C at this position. The edited form (7SL I) was found preferentially in the cytoplasm, and the pre-edited form in the nucleus. 7SL I is mainly bound to ribosomes, whereas 7SL II is more abundant in ribosome-free particles. Mutations introduced in regions outside the editing site were found to occur in a single conformation, suggesting that the editing event is not the only factor that determines the conformation of the molecule. This study is the first description of an editing event on a small RNA other than tRNA and is the first report of C --> U editing in trypanosomes. We propose a novel role for RNA editing in controlling the conformation of the 7SL RNA in vivo.

Characterization of a novel trypanosomatid small nucleolar RNA.

Trypanosomes possess unique RNA processing mechanisms including trans- splicing of pre-mRNA and RNA editing of mitochondrial transcripts. The previous finding of a trimethylguanosine (TMG) capped U3 homologue in trypanosomes suggests that rRNA processing may be related to the processing in other eukaryotes. In this study, we describe the first trypanosomatid snoRNA that belongs to the snoRNAs that were shown to guide ribose methylation of rRNA. The RNA, identified in the monogenetic trypanosomatid Leptomonas collosoma, was termed snoRNA-2 and is encoded by a multi-copy gene. SnoRNA-2 is 85 nt long, it lacks a 5' cap and possesses the C and D boxes characteristic to all snoRNAs that bind fibrillarin. Computer analysis indicates a potential for base-pairing between snoRNA-2 and 5.8S rRNA, and 18S rRNA. The putative interaction domains obey the rules suggested for the interaction of guide snoRNA with its rRNA target for directing ribose methylation on the rRNA. However, mapping the methylated sites on the 5.8S rRNA and 18S rRNA indicates that the expected site on the 5.8S is methylated, whereas the site on the 18S is not. The proposed interaction with 5.8S rRNA is further supported by the presence of psoralen cross-link sites on snoRNA-2. GenBank search suggests that snoRNA-2 is not related to any published snoRNAs. Because of the early divergence of the Trypanosomatidae from the eukaryotic lineage, the presence of a methylating snoRNA that is encoded by a multi-copy gene suggests that methylating snoRNAs may have evolved in evolution from self-transcribed genes.

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: the Leptomonas collosoma U5 RNA and its coding gene.

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5' phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016-4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6.U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem-loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNACys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5' splice site region (positions -1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.

The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression.

We have previously reported the co-purification of a tRNA-like molecule with the Trypanosoma brucei SRP complex [Béjà et al . (1993) Mol. Biochem. Parasitol . 57, 223-230]. To examine whether the trypanosome SRP has a unique composition compared with that of other eukaryotes, we analyzed the 7SL RNA and the SRP complex of the monogenetic trypanosomatid Leptomonas collosoma. The 7SL RNA from L. collosoma was cloned, and its gene was sequenced. In contrast to T. brucei , two 7SL RNA transcripts were detected in L.collosoma that originate from a single-copy gene. Using stable cell lines expressing tagged 7SL RNA, we demonstrate that the tRNAArggene located 98 bp upstream to the 7SL RNA serves as part of the 7SL RNA extragenic promoter. The steady-state level of 7SL RNA was found to be tightly regulated, since the presence of the gene on the multi-copy plasmid repressed the synthesis of the chromosomal gene. Cell lines carrying truncated 7SL RNA genes were established and their expression indicated that domain I is essential for expressing the 7SL RNA. No constructs carrying portions of the 7SL RNA were expressed, except for a construct which lacked 23 nt from the 3'end of the RNA. This suggests that 90% of the 7SL RNA molecule is important for its maintenance as a stable small RNA. We propose that the repression phenomenon may originate from a regulatory mechanism that coordinates the level of the 7SL RNA by its binding proteins.

Preferential release of catecholamine from permeabilized PC12 cells by alpha- and beta-type protein kinase C subspecies.

Protein kinase C (PKC) is now recognized as comprising two groups of closely related subspecies. The PKC gamma enzyme is apparently present only in central nervous tissues, and hence was expected to participate in neurotransmitter release. We have utilized a 'depletion-insertion' method to identify the PKCs participating in the exocytotic response. PC12 cells were 'down-regulated' by prior treatment (24 h) with phorbol 12-myristate 13-acetate (PMA; 1 microM), which nearly abolished endogenous PKC activity. Down-regulated PC12 cells were loaded with [3H]dopamine, permeabilized with digitonin, and recombinant or purified PKCs were inserted and activated with a low dose of PMA (20 nM). Among group A PKCs, PKC alpha was the most effective activator of [3H]dopamine release (215%), followed by beta II (185%) and beta I (150%). PKC gamma had no consistent effect on neurotransmitter release. PC12 cells express PKC alpha and PKC beta, but not PKC gamma, as revealed by Northern-blot analysis. We therefore postulate that PKC alpha and PKC beta participate in neurotransmitter release, whereas PKC gamma might be involved in other neuronal functions.