Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation.

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.

RETRACTED: A 5' fragment of Xist can sequester RNA produced from adjacent genes on chromatin.

Xist requires Repeat-A, a protein-binding module in its first two kilobases (2kb), to repress transcription. We report that when expressed as a standalone transcript in mouse embryonic stem cells (ESCs), the first 2kb of Xist (Xist-2kb) does not induce transcriptional silencing. Instead, Xist-2kb sequesters RNA produced from adjacent genes on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription and can be induced by lncRNAs with similar sequence composition to Xist-2kb. We did not detect sequestration by full-length Xist, but we did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. Xist-2kb associated with SPEN, a Repeat-A binding protein required for Xist-induced transcriptional silencing, but SPEN was not necessary for sequestration. Thus, when expressed in mouse ESCs, a 5' fragment of Xist that contains Repeat-A sequesters RNA from adjacent genes on chromatin and associates with the silencing factor SPEN, but it does not induce transcriptional silencing. Instead, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. We propose that sequestration is mechanistically related to the Repeat-A dependent stabilization and tethering of Xist near actively transcribed regions of chromatin.

Upregulation of LINC00319 indicates a poor prognosis and promotes cell proliferation and invasion in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (CSCC), an epidermal keratinocyte-derived skin tumor, is one of the most leading causes of cancer-associated morbidity and mortality worldwide. Long noncoding RNAs have emerged as key regulators of tumor development and progression. Recent studies have identified LINC00319, a long intergenic noncoding RNA, as an oncogene in lung cancer. However, the biological role of LINC00319 in CSCC remains largely unknown. The current study aimed to explore the role of LINC00319 in CSCC and uncover the molecular mechanisms. In current study, we found that LINC00319 was significantly upregulated in both CSCC tissues and cell lines. Besides, the χ2 test showed that increased expression of LINC00319 was associated with larger tumor size, advanced TNM stage, and lymphovascular invasion. Gain-of-function and loss-of-function approaches were applied to investigate the effects of LINC00319 on CSCC cells. Functional studies demonstrated that LINC00319 promoted CSCC cell proliferation, accelerated cell cycle progression, facilitated cell migration and invasion, and inhibited cell apoptosis. Mechanistic studies revealed that LINC00319 exerts its oncogenic functions in CSCC via miR-1207-5p-mediated regulation of cyclin-dependent kinase 3. Taken together, upregulation of LINC00319 implies a potential link with poor prognosis and reflects CSCC progression. Collectively, this study may provide some evidence for LINC00319 as a candidate target in CSCC treatment.

Synthetic artificial "long non-coding RNAs" targeting oncogenic microRNAs and transcriptional factors inhibit malignant phenotypes of bladder cancer cells.

Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play important roles in human cancers, acting as transcriptional and post-transcriptional regulators, respectively. These phenomena raise questions about the ability of an artificial device to simultaneously regulate miRNAs and TFs. In this study, we aimed to construct artificial long non-coding RNAs, "alncRNAs", and to investigate their therapeutic effects on bladder cancer cell lines. Based on engineering principles of synthetic biology, we combined tandem arrayed aptamer cDNA sequences for TFs with tandem arrayed cDNA copies of binding sites for the miRNAs to construct alncRNAs. In order to prove the utility of this platform, we chose ß-catenin and the miR-183-182-96 cluster as the functional targets and used the bladder cancer cell lines 5637 and SW780 as the test models. Dual-luciferase reporter assay, real-time quantitative PCR (qRT-PCR) and related phenotypic experiments were used to test the expression of related genes and the therapeutic effects of our devices. The result of dual-luciferase reporter assay and qRT-PCR showed that alncRNAs could inhibit transcriptional activity of TFs and expression of corresponding microRNAs. Using functional experiments, we observed decreased cell proliferation, increased apoptosis, and motility inhibition in alncRNA-infected bladder cancer cells. What's more, follow-up mechanism experiments further confirmed the anti-tumor effect of our devices. In summary, our synthetic devices indeed function as anti-tumor regulators, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cells. Most importantly, anti-cancer effects were induced by the synthetic alncRNAs in the bladder cancer lines. Our devices, all in all, provided a novel strategy and methodology for cancer studies, and might show a great potential for cancer therapy if the challenges of in vivo DNA delivery are overcome.

Xist imprinting is promoted by the hemizygous (unpaired) state in the male germ line.

The long noncoding X-inactivation-specific transcript (Xist gene) is responsible for mammalian X-chromosome dosage compensation between the sexes, the process by which one of the two X chromosomes is inactivated in the female soma. Xist is essential for both the random and imprinted forms of X-chromosome inactivation. In the imprinted form, Xist is paternally marked to be expressed in female embryos. To investigate the mechanism of Xist imprinting, we introduce Xist transgenes (Tg) into the male germ line. Although ectopic high-level Xist expression on autosomes can be compatible with viability, transgenic animals demonstrate reduced fitness, subfertility, defective meiotic pairing, and other germ-cell abnormalities. In the progeny, paternal-specific expression is recapitulated by the 200-kb Xist Tg. However, Xist imprinting occurs efficiently only when it is in an unpaired or unpartnered state during male meiosis. When transmitted from a hemizygous father (+/Tg), the Xist Tg demonstrates paternal-specific expression in the early embryo. When transmitted by a homozygous father (Tg/Tg), the Tg fails to show imprinted expression. Thus, Xist imprinting is directed by sequences within a 200-kb X-linked region, and the hemizygous (unpaired) state of the Xist region promotes its imprinting in the male germ line.