Circular IRE-type RNAs of the NR5A1 gene are formed in adrenocortical cells.

The recently discovered circular RNAs (circRNAs) are mostly formed by back-splicing where the downstream 5' splice site splices to the upstream 3' splice site by conventional pre-mRNA splicing. These circRNAs regulate gene expression by acting as sponges for micro-RNAs or RNA-binding proteins. Here we show that the NR5A1 (previously called Ad4BP or SF-1) gene which is exclusively expressed in the adrenal cortex and steroidogenic tissue can form atypical circRNAs by unconventional splicing. Two stem loops with inositol-requiring protein-1α (IRE1α) cleavage sites are connected by an IRE1α cleavage site to form a circRNA (circIRE RNA). From total RNA of normal human adrenal cortex, we detected a circIRE RNA with connected ends by IRE1α cleavage sites in exon 6 and exon 1 (circIRE NR5A1 ex6-1 RNA). circIRE NR5A1 ex6-1 RNA was not detected in the adrenocortical cancer cell line, H295R. When IRE1α was expressed in H295R cells a different circIRE NR5A1 RNA connecting IRE1-cleavage sites in exon 7 and exon 1 was detected (circIRE NR5A1 ex7-1 RNA). The expression of this circIRE RNA was inhibited by the IRE1 inhibitor 1, STF-083010, implicating that it was formed via the ER stress pathway, where IRE1α is a major factor. This is the first report of this type of circular RNA connected by IRE1-cleavage sites found to be expressed in mammalian cells in a tissue-specific manner. To our surprise, the concomitant expression of NR5A1 was increased by IRE1α implicating that NR5A1 was not subjected to IRE1-dependent decay of mRNA (RIDD) but rather activating a transcriptional regulatory network to cope with ER stress in steroidogenic tissue reminiscent to XBP1 in other tissue. We believe this is the first report of such tissue-specific transcriptional cascade responding to ER stress as well as the novel finding of circular RNAs connected by IRE1α cleavage sites expressed in mammalian tissue.

HMGA1a Induces Alternative Splicing of the Estrogen Receptor-αlpha Gene by Trapping U1 snRNP to an Upstream Pseudo-5' Splice Site.

Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells. Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2'-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a. RNA-binding of HMGA1a was checked by RNA-EMSA. In vitro splicing assay was performed to check the direct involvement of HMGA1a in splicing regulation. RNA-EMSA assay in the presence of purified U1 snRNP was performed with psoralen UV crosslinking to check complex formation of HMGA1a-U1 snRNP at the upstream pseudo-5' splice site of exon 1. Results: HMGA1a induced exon skipping of a shortened exon 1 of ERα in in vitro splicing assays that was blocked by the HMGA1a RNA decoy and sequence-specific RNA-binding was confirmed by RNA-EMSA. RNA-EMSA combined with psoralen UV crosslinking showed that HMGA1a trapped purified U1 snRNP at the upstream pseudo-5' splice site. Conclusions: Regulation of ERα alternative splicing by an HMGA1a-trapped U1 snRNP complex at the upstream 5' splice site of exon 1 offers novel insight on 5' splice site regulation by U1 snRNP as well as a promising target in breast cancer therapy where alternative splicing of ERα is involved.

HMGA1a induces alternative splicing of estrogen receptor alpha in MCF-7 human breast cancer cells.

The high-mobility group A protein 1a (HMGA1a) protein is known as an oncogene whose expression level in cancer tissue correlates with the malignant potential, and known as a component of senescence-related structures connecting it to tumor suppressor networks in fibroblasts. HMGA1 protein binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. Our previous studies have shown that sequence-specific RNA-binding of HMGA1a induces exon-skipping of Presenilin-2 exon 5 in sporadic Alzheimer disease. Here we show that HMGA1a induced exon-skipping of the estrogen receptor alpha (ERα) gene and increased ERα46 mRNA expression in MCF-7 breast cancer cells. An RNA-decoy of HMGA1a efficiently blocked this event and reduced ERα46 protein expression. Blockage of HMGA1a RNA-binding property consequently induced cell growth through reduced ERα46 expression in MCF-7 cells and increased sensitivity to tamoxifen in the tamoxifen-resistant cell line, MCF-7/TAMR1. Stable expression of an HMGA1a RNA-decoy in MCF-7 cells exhibited decreased ERα46 protein expression and increased estrogen-dependent tumor growth when these cells were implanted in nude mice. These results show HMGA1a is involved in alternative splicing of the ERα gene and related to estrogen-related growth as well as tamoxifen sensitivity in MCF-7 breast cancer cells.

The significance of anti-Müllerian hormone concentration in seminal plasma for spermatogenesis.

BACKGROUND: The function of anti-Müllerian hormone (AMH) in seminal plasma in adulthood is uncertain. We examined the significance of seminal AMH for spermatogenesis. METHODS: We measured seminal concentrations of AMH in 39 oligozoospermic men (mean age +/- SD, 32.7 +/- 4.3 years) and 10 normal volunteers to examine the association of seminal AMH with spermatogenesis. The seminal concentrations of AMH in oligozoospermic men (149.3 +/- 254.0 pmol/l) were significantly lower than in normal men (249.0 +/- 167.7 pmol/l; P +/- 0.0337). Seminal AMH concentration correlated significantly with sperm concentration (r = 0.339, P = 0.0350) and mean testicular volume (r = 0.440, P = 0.246). The serum concentration of LH (r = - 0.365, P = 0.0241), but not FSH, testosterone or estradiol, correlated significantly with AMH concentration in seminal plasma. CONCLUSIONS: AMH in seminal plasma may be important for sperm production, and is a good marker for Sertoli cell development.

Therapeutic strategy after microsurgical varicocelectomy in the modern assisted reproductive technology era.

In this study, we searched for prognostic factors at preoperative examination for the improvement in spermatogenesis of patients undergoing varicocelectomy. Eighty patients with varicocele testis underwent microsurgical varicocelectomy. Before surgery, the seminogram, testicular volume, varicocele grade, and serum FSH, LH, testosterone, prolactin, and estradiol were evaluated. Postoperatively, semen analysis was performed every 3 months. We assessed the associations between the preoperative variables and postoperative seminogram improvement. 0f 80 patients, 37 showed improvement, usually by 6 months. Patient age, duration of sterility, testicular volume, sperm motility, morphology, semen volume, serum LH, testosterone, prolactin, and estradiol showed little difference between responders and non-responders. A small left testis, or a grade III varicocele decreased the likelihood of improvement. Patients with a sperm count of 10-20 x 10(6)/ml were significantly more likely to respond to varicocelectomy than those with sperm counts <5 x 10(6)/ml. Patients with elevated FSH were less likely to respond, as were those with a Johnsen score below 6. Varicocelectomy alone is unlikely to improve sperm counts of patients with a sperm count below 5 x 10(6)/ml, high FSH, small left testes, or Johnsen scores below 6. In conclusion, for couples in this situation, assisted reproductive technology coupled with varicocelectomy should be proposed.