Researchers Identify Proteins Required for X Chromosome Silencing by the Long Noncoding RNA Xist

A recent paper identified a group of proteins that interact directly with the long noncoding RNA (lncRNA) Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals.

Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides. This somewhat arbitrary limit distinguishes lncRNAs from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs. LncRNAs have been found to be involved in numerous biological roles including imprinting, epigenetic gene regulation, cell cycle and apoptosis, and metastasis and prognosis in solid tumors. Most lncRNAs are expressed only in a few cells rather than whole tissues, or they are expressed at very low levels, making them difficult to study.

The lncRNA Xist is essential to the process of silencing one copy of the X chromosome in female embryos. Having two copies of the X chromosome is an abnormality that leads to death early of the embryo during development.

Investigators at the California Institute of Technology (Pasadena, CA, USA) developed a technique to identify those proteins that naturally interact with Xist in the cell. They combined RNA antisense purification with mass spectrometry (RAP-MS). The investigators used the antisense purification technique to extract and purify Xist lncRNA molecules, as well as the proteins that directly interact with Xist, from mouse embryonic stem cells. Then, they employed quantitative mass spectrometry to identify those interacting proteins.

They reported in the April 27, 2015, online edition of the journal Nature that 10 proteins were specifically associated with Xist. Three of these proteins—SAF-A (Scaffold attachment factor-A), LBR (Lamin B Receptor), and SHARP (SMRT and HDAC associated repressor protein)—were required for Xist-mediated transcriptional silencing. Further analysis revealed that the direct interaction of Xist and SHARP triggered a series of steps that led to the exclusion of RNA polymerase II from the cell's DNA, thus preventing transcription and gene expression.

Senior author Dr. Mitchell Guttman, assistant professor of biology and biological engineering at the California Institute of Technology, said, "To start to make sense of what makes lncRNAs special and how they can control all of these different cellular processes, we need to be able to understand the mechanism of how any lncRNA gene can work. Because Xist is such an important molecule and because so much is known about what it does, it seemed like a great system to try to dissect the mechanisms of how it and other lncRNAs work. We are starting to pick apart how lncRNAs work. We now know, for example, how Xist localizes to sites on X, how it silences transcription, and how it can change DNA structure. One of the things that is really exciting for me is that we can potentially leverage the principles used by lncRNAs, move them around in the genome, and use them as therapeutic agents to target specific defective pathways in disease."

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California Institute of Technology