Molecular Mechanisms of Alu and LINE-1 Interspersed Repetitive Sequences Reveal Diseases of Visual System Dysfunction.

BACKGROUND: Short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINE-1s) are the abundant and well-characterized repetitive elements in the human genome. METHODS: For this review, all relevant original research studies were assessed by searching electronic databases, including PubMed, Google Scholar, and Web of Science, by using relevant keywords. Accumulating evidence indicates that the disorder of gene expression regulated by these repetitive sequences is one of the causes of the diseases of visual system dysfunction, including retinal degenerations, glaucoma, retinitis punctata albescens, retinitis pigmentosa, geographic atrophy, and age-related macular degeneration, suggesting that SINEs and LINE-1s may have great potential implications in ophthalmology. RESULTS: Alu elements belonging to the SINEs are present in more than one million copies, comprising 10% of the human genome. CONCLUSION: This study offers recent advances in Alu and LINE-1 mechanisms in the development of eye diseases. The current study could advance our knowledge of the roles of SINEs and LINE-1s in the developing process of eye diseases, suggesting new diagnostic biomarkers, therapeutic strategies, and significant points for future studies.

This study reveals the Alu and LINE-1 interspersed repetitive sequences involved in the diseases of visual system dysfunction.This study shows the disorder of gene expression regulated by SINEs and LINE-1s sequences is one of the causes of the diseases of visual system dysfunction.This study suggests recent advances in Alu and LINE-1 mechanisms are involved in eye diseases.

Anti-aging and anti-oxidant activities of murine short interspersed nuclear element antisense RNA.

Short interspersed nuclear elements (SINEs) play a key role in regulating gene expression, and SINE RNAs are involved in age-related diseases. We investigated the anti-aging effects of a genetically engineered murine SINE B1 antisense RNA (B1as RNA) and explored its mechanism of action in naturally senescent BALB/c (≥14 months) and moderately senscent C57BL/6N (≥9 months) mice. After tail vein injection, B1as RNA was available in the blood of mice for approximately 30 min, persisted for approximately 2-4 h in most detected tissues and persisted approximately 48 h in lungs. We found that treatment with B1as RNA improved stamina and promoted hair re-growth in aged mice. Treatment with B1as RNA also partially rescued the increase in mitochondrial DNA copy number in liver and spleen tissues observed in aged and moderately senescent mice. Finally, treatment with B1as RNA increased the activities of superoxide dismutase and glutathione peroxidase in aged and moderately senescent mice, reduced these animals' malondialdehyde and reactive oxygen species levels, and modulated the expression of several aging-associated genes, including Sirtuin 1, p21, p16Ink4a, p15Ink4b and p19Arf, and anti-oxidant genes (Sesn1 and Sesn 2). These data suggest that B1as RNA inhibits the aging process by enhancing antioxidant activity, promoting the scavenging of free radicals, and modulating the expression of aging-associated genes. This is the first report describing the anti-aging activity of SINE antisense RNA, which may serve as an effective nucleic acid drug for the treatment of age-related diseases.

Preparation of genetically engineered murine SINE RNA without endotoxin contamination.

RNAs have been elucidated to play the critical role in regulating gene expression and to be expected as effective drugs in the treatment of cancer and age-related diseases. RNAs are extracted by SDS-NaCl centrifugation after transformation of E.coli by expression vectors, which is a method to obtain genetically engineered RNAs. But the prepared RNAs by this method contain endotoxin, which limits their application in vivo and in cell experments. Here we improved SDS-NaCl filtration method based on SDS-NaCl centrifugation method. Endotoxin removal efficiency of SDS-NaCl filtration was nearly 4.2 times more than did SDS-NaCl centrifugation. Triton X-114 phase separation was used to reduce futher the endotoxin content of SDS-NaCI filtration-extracted RNA (from 11.25 EU/µg RNA/ml to 0.08 EU/µg RNA/ml). RNA prepared using the methods established in this paper meets the requirements for in vivo and cell culture experiments. Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3 E. coli (DE3 transformed by pET-B1as expression vector which containing a tandem SINE B1 elements) using SDS-NaCl filtration incorporating Triton X-114 phase separation.•The endotoxin removal efficiency of SDS-NaCl filtration is higher than that of SDS-NaCl centrifugation.•RNA prepared by SDS-NaCl filtration incorporating Triton X-114 meets the requirements for in vivo experiments on animals.

The preparation of endotoxin-free genetically engineered murine B1 antisense RNA.

One of the major limitations in the production of genetically engineered RNA from Escherichia coli (E. coli) is contamination by endotoxin. Here we report the first method that is capable of removing endotoxin from genetically engineered RNA. As a proof of concept, we transformed E. coli with a plasmid containing a tandem short interspersed nuclear elements from the mouse genome (SINE B1 elements). We then evaluated several extraction methods (SDS-NaCl centrifugation, SDS-NaCl filtration, TRIzol and SDS hot-phenol) and refinements thereof, and measured the resulting RNA yield, RNA purity, RNA integrity and endotoxin content. SDS-NaCl filtration with 2 mol/L NaCl, incorporating DEPC as an RNA protective agent, effectively removed endotoxin and resulted in a good RNA yield. Triton X-114 phase separation further reduced the endotoxin content of SDS-NaCl filtration-extracted RNA. RNA extracted by SDS-NaCl filtration with Triton X-114 phase separation did not cause adverse reactions in BALB/c mice and did not induce fever in rabbits when injected into these animals. The RNA met the requirements of nucleic acid reagents for in vivo experiments on animals.