Decoding the Functional Role of Fast, a Long Non-Coding RNA Transcribed Antisense to Nr5a1.

Abstract

Over the past decade, research on long non-coding RNAs implicates them in regulatory roles associated with gene expression and chromatin architecture. While searching for conserved regulatory elements, an expressed sequence tag lacking an identifiable open reading frame was identified associated with Ftz-f1 (Nr5a1), the gene encoding steroidogenic factor 1 (SF-1). The nuclear receptor SF-1 is a key determinant and regulator of the adrenal and reproductive axes. Mammals with SF-1 mutations display a range of phenotypes, including absence of adrenal glands and gonads, diminished pituitary gonadotropins, disruption of the ventromedial hypothalamus, ovarian failure, and XY-sex reversal. While there are numerous studies on the function and regulation of SF-1, none involved mechanisms that require a noncoding RNA (ncRNA). Identification of the Ftz-F1 associated transcript, or Fast, and its association with SF-1 suggested a role for the ncRNA in SF-1 expression and/or activity and prompted further investigation of Fast. Directional RT-PCR was used to determine transcriptional orientation of Fast and demonstrated it is transcribed in the opposite orientation of Ftz-F1. Fast full-length structure was delineated using 5' and 3' RACE and sequence analysis of amplified Fast cDNA. This showed Fast has three exons and is polyadenylated and derived from multiple transcriptional start sites that border and extend into exon 1g of Ftz-F1. In addition to identification of a third exon, sequence analysis revealed two amplified, possible spliced variants that lacked part of exon 3. RT-PCR analysis of RNA from multiple mouse tissues and endocrine cell lines showed Fast and SF-1 share the same expression profile. To help define the sub-cellular location of Fast, nuclear and cytoplasmic RNA was isolated from MA-10 cells, a Leydig cell line, and analyzed for the presence of Fast by RT-PCR. This revealed that Fast is predominantly located in the cytoplasm. The similar tissue expression pattern for SF-1 and Fast suggested they share regulatory mechanisms. Further investigation showed that P-19 embryonal-carcinoma cells treated with retinoic acid had decreased SF-1 and Fast transcript levels and that granulosa cells isolated from mice treated with pregnant mare's serum gonadotropin had elevated levels of both Fast and SF-1 mRNA. Together, these findings indicate that Fast is a long non-coding RNA that shares the same tissue-restricted expression profile and hormone regulation as SF-1, suggesting it acts within a similar biological pathway. We hypothesize that Fast acts either in conjunction with SF-1 and thus is required for proper development and function of adrenal glands and gonads.