Differential expression of nasal embryonic LHRH factor (NELF) variants in immortalized GnRH neuronal cell lines

Abstract

NELF, a protein identified in migratory GnRH neurons, is predominantly nuclear and alternatively spliced. However, specific NELF splice variants expressed in immortalized GnRH neuronal cell lines from mouse and human are not known. RNA from migratory (GN11 and NLT) and postmigratory (GT1-7) cells in mouse, and (FNCB4-hTERT) cells in human was subjected to RT-PCR. RT-PCR products were cloned, electrophoresed on denaturing gradient gels and sequenced. In addition, quantitative RT-PCR was performed using variant-specific primers. Western blot and immunofluorescence using confocal microscopy were performed for selected variants. Nelf variant 2 (v2), which contains a nuclear localization signal (NLS), was the predominant variant in all mouse and human GnRH neurons. Variants without a NLS (v3 in mouse; v4 in human) were identified. In mouse, v2 protein expression was nuclear, while v3 was non-nuclear. In mouse GnRH neurons, six Nelf splice variant transcripts were identified, including three previously unreported variants. In human, four NELF variant transcripts were observed. In both mouse and human, nuclear and non-nuclear variant transcript and protein were identified, explaining variable NELF cellular localization.

Nasal Placode Development, GnRH Neuronal Migration and Kallmann Syndrome

“…However, in a second Nsmf KO model, no changes in GnRH neuronal number or reproductive function was observed (Spilker et al, 2016). To date, six variant transcripts of Nsmf have been identified (Quaynor et al, 2014). Further studies are needed to identify whether splice variant expression might account for these differences.…”

Jacob, a Synapto-Nuclear Protein Messenger Linking N-methyl-D-aspartate Receptor Activation to Nuclear Gene Expression

“…The Jacob/NSMF gene structure is rather complex with 16 exons (Figure 2A) and high sequence conservation between mammalian species. It has been shown that at least 5 out of 16 exons of the Jacob/NSMF gene can be alternatively spliced (Figure 2A; Miura et al, 2004;Dieterich et al, 2008;Kindler et al, 2009;Miura et al, 2013;Quaynor et al, 2014;Joglekar et al, 2021). The existence of many Jacob/NSMF isoforms are predicted, but not all of them are experimentally confirmed and not much information is available about brain region-and cell type-specific expression of splice variants and regulation of expression in development.…”