Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS is characterized by hyperandrogenism and ovulatory dysfunction. Women with PCOS have a high prevalence of obesity, insulin resistance (IR), increased blood pressure (BP), and activation of the renin angiotensin system (RAS). Effective evidence-based therapeutics to ameliorate the cardiometabolic complications in PCOS are lacking. The sodium-glucose cotransporter-2 (SGLT2) inhibitor Empagliflozin (EMPA) reduces BP and hyperglycemia in type 2 diabetes mellitus. We hypothesized that hyperandrogenemia upregulates renal SGLT2 expression and that EMPA ameliorates cardiometabolic complications in a hyperandrogenemic PCOS model. Four-week-old female Sprague Dawley rats were treated with dihydrotestosterone (DHT) for 90 days, and EMPA was co-administered for the last three weeks. DHT upregulated renal SGLT2, SGLT4, and GLUT2, but downregulated SGLT3 mRNA expression. EMPA decreased DHT-mediated increases in fat mass, plasma leptin, and BP, but failed to decrease plasma insulin, HbA1c, or albuminuria. EMPA decreased DHT-mediated increase in renal angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE2), and angiotensin II type 1 receptor (AGT1R) mRNA and protein expression. In summary, SGLT2 inhibition proved beneficial in adiposity and BP reduction in a hyperandrogenemic PCOS model; however, additional therapies may be needed to improve IR and renal injury.

Small Cajal body-specific RNAs (scaRNAs) are part of small Cajal body-specific ribonucleoproteins (scaRNPs) that modify small nuclear RNA (snRNA) in Cajal bodies (CBs). Several scaRNAs (scaRNA 2, 9 and 17) have been found to generate smaller, nucleolus-enriched fragments. We hypothesize that the fragments derived from scaRNA 2, 9 and 17 form regulatory RNPs that influence the level of modifications within rRNA by altering small nucleolar RNP (snoRNP) activity. Here we show that external factors such as DNA damaging agents can alter the scaRNA9 full length to processed fragment ratio. We also show that full-length scaRNA2 levels are likewise impacted by DNA damage, which correlates with the disruption of SMN, coilin and WRAP53 co-localization in CBs. The dynamics of scaRNA9 were also shown to be affected by Drosha levels, which suggests that this protein may participate in the biogenesis and processing of this non-coding RNA. Identification of factors that contribute to scaRNA 2, 9 and 17 processing may facilitate an assessment of how external stress can lead to changes in rRNA modifications.

Our results demonstrate that Cajal bodies and the microRNA (miRNA) processing machinery functionally interact and together contribute to the biogenesis of miRNAs and small nuclear ribonucleoproteins.

Ribosomes can be heterogeneous, and the major contributor to ribosome heterogeneity is variation in rRNA modification. There are two major types of rRNA modification, pseudouridylation and ribose methylation. In humans, the majority of these rRNA modifications are conducted by two classes of small nucleolar ribonucleoproteins (snoRNPs), which contain a guide RNA (small nucleolar RNA, snoRNA) complexed with proteins. Box H/ACA snoRNPs conduct pseudouridylation modifications and box C/D snoRNPs generate ribose methylation modifications. It is unclear how ribosome heterogeneity is accomplished in regards to the understanding of the signals and factors that regulate rRNA modifications. We have recently reported that a new class of RNP, that we term regulatory RNP (regRNP), may contribute to rRNA modification as well as the modification of nucleolar trafficked U6 snRNA, via interactions with snoRNPs. Here we report the identification of additional regRNP activities that influence the methylation of two sites within 18S rRNA, two sites within 28S rRNA and one site within U6 snRNA. These findings provide additional proof that regulation of snoRNP activity contributes to ribosome heterogeneity.

MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is comprised of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal Body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of co-immunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.