Nitric oxide and sickle cell disease-Is there a painful connection?

Sickle cell disease is the most common hemoglobinopathy and affects millions worldwide. The disease is associated with severe organ dysfunction, acute and chronic pain, and significantly decreased life expectancy. The large body of work demonstrating that hemolysis results in rapid consumption of the endogenous vasodilator nitric oxide, decreased nitric oxide production, and promotion of vaso-occlusion provides the basis for the hypothesis that nitric oxide bioavailability is reduced in sickle cell disease and that this deficit plays a role in sickle cell disease pain. Despite initial promising results, large clinical trials using strategies to increase nitric oxide bioavailability in sickle cell disease patients yielded no significant change in duration or frequency of acute pain crises. Further, recent investigations showed that sickle cell disease patients and mouse models have elevated baseline levels of blood nitrite, a reservoir for nitric oxide formation and a product of nitric oxide metabolism, regardless of pain phenotype. These conflicting results challenge the hypotheses that nitric oxide bioavailability is decreased and that it plays a significant role in the pathogenesis in sickle cell disease acute pain crises. Conversely, a large body of work demonstrates that nitric oxide, as a neurotransmitter, has a complex role in pain neurobiology, contributes to the development of central sensitization, and can mediate hyperalgesia in inflammatory and neuropathic pain. These results support an alternative hypothesis: one proposing that altered nitric oxide signaling may contribute to the development of neuropathic and/or inflammatory pain in sickle cell disease through its role as a neurotransmitter.

Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.