We describe the identification and characterization of circular intronic long noncoding RNAs in human cells, which accumulate owing to a failure in debranching. The formation of such circular intronic RNAs (ciRNAs) can be recapitulated using expression vectors, and their processing depends on a consensus motif containing a 7 nt GU-rich element near the 5' splice site and an 11 nt C-rich element close to the branchpoint site. In addition, we show that ciRNAs are abundant in the nucleus and have little enrichment for microRNA target sites. Importantly, knockdown of ciRNAs led to the reduced expression of their parent genes. One abundant such RNA, ci-ankrd52, largely accumulates to its sites of transcription, associates with elongation Pol II machinery, and acts as a positive regulator of Pol II transcription. This study thus suggests a cis-regulatory role of noncoding intronic transcripts on their parent coding genes.

Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell-derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

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