Decoding protein-RNA interactions using CLIP-based methodologies.

Protein-RNA interactions are central to all RNA processing events, with pivotal roles in the regulation of gene expression and cellular functions. Dysregulation of these interactions has been increasingly linked to the pathogenesis of human diseases. High-throughput approaches to identify RNA-binding proteins and their binding sites on RNA - in particular, ultraviolet crosslinking followed by immunoprecipitation (CLIP) - have helped to map the RNA interactome, yielding transcriptome-wide protein-RNA atlases that have contributed to key mechanistic insights into gene expression and gene-regulatory networks. Here, we review these recent advances, explore the effects of cellular context on RNA binding, and discuss how these insights are shaping our understanding of cellular biology. We also review the potential therapeutic applications arising from new knowledge of protein-RNA interactions.

Targeted inhibition of kisspeptin neurons reverses hyperandrogenemia and abnormal hyperactive LH secretion in a preclinical mouse model of polycystic ovary syndrome.

STUDY QUESTION: Do hyperactive kisspeptin neurons contribute to abnormally high LH secretion and downstream hyperandrogenemia in polycystic ovary syndrome (PCOS)-like conditions and can inhibition of kisspeptin neurons rescue such endocrine impairments? SUMMARY ANSWER: Targeted inhibition of endogenous kisspeptin neuron activity in a mouse model of PCOS reduced the abnormally hyperactive LH pulse secretion and hyperandrogenemia to healthy control levels. WHAT IS KNOWN ALREADY: PCOS is a reproductive disorder characterized by hyperandrogenemia, anovulation, and/or polycystic ovaries, along with a hallmark feature of abnormal LH hyper-pulsatility, but the mechanisms underlying the endocrine impairments remain unclear. A chronic letrozole (LET; aromatase inhibitor) mouse model recapitulates PCOS phenotypes, including polycystic ovaries, anovulation, high testosterone, and hyperactive LH pulses. LET PCOS-like females also have increased hypothalamic kisspeptin neuronal activation which may drive their hyperactive LH secretion and hyperandrogenemia, but this has not been tested. STUDY DESIGN, SIZE, DURATION: Transgenic KissCRE+/hM4Di female mice or littermates Cre- controls were treated with placebo, or chronic LET (50 µg/day) to induce a PCOS-like phenotype, followed by acute (once) or chronic (2 weeks) clozapine-N-oxide (CNO) exposure to chemogenetically inhibit kisspeptin cells (n = 6 to 10 mice/group). PARTICIPANTS/MATERIALS, SETTING, METHODS: Key endocrine measures, including in vivo LH pulse secretion patterns and circulating testosterone levels, were assessed before and after selective kisspeptin neuron inhibition and compared between PCOS groups and healthy controls. Alterations in body weights were measured and pituitary and ovarian gene expression was determined by qRT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Acute targeted inhibition of kisspeptin neurons in PCOS mice successfully lowered the abnormally hyperactive LH pulse secretion (P < 0.05). Likewise, chronic selective suppression of kisspeptin neuron activity reversed the previously high LH and testosterone levels (P < 0.05) down to healthy control levels and rescued reproductive gene expression (P < 0. 05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ovarian morphology was not assessed in this study. Additionally, mouse models can offer mechanistic insights into neuroendocrine processes in PCOS-like conditions but may not perfectly mirror PCOS in women. WIDER IMPLICATIONS OF THE FINDINGS: These data support the hypothesis that overactive kisspeptin neurons can drive neuroendocrine PCOS-like impairments, and this may occur in PCOS women. Our findings complement recent clinical investigations using NKB receptor antagonists to lower LH in PCOS women and suggest that pharmacological dose-dependent modulation of kisspeptin neuron activity may be a valuable future therapeutic target to clinically treat hyperandrogenism and lower elevated LH in PCOS women. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by NIH grants R01 HD111650, R01 HD090161, R01 HD100580, P50 HD012303, R01 AG078185, and NIH R24 HD102061, and a pilot project award from the British Society for Neuroendocrinology. There are no competing interests.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.