Long noncoding RNA and protein abundance in lncRNPs.

Although long noncoding RNAs (lncRNAs) are generally expressed at low levels, emerging evidence has revealed that many play important roles in gene regulation by a variety of mechanisms as they engage with proteins. Given that the abundance of proteins often greatly exceeds that of their interacting lncRNAs, quantification of the relative abundance, or even the exact stoichiometry in some cases, within lncRNA-protein complexes is helpful for understanding of the mechanism(s) of action of lncRNAs. We discuss methods used to examine lncRNA and protein expression at the single cell, subcellular, and suborganelle levels, the average and local lncRNA concentration in cells, as well as how lncRNAs can modulate the functions of their interacting proteins even at a low stoichiometric concentration.

MS2-MBP-Based Affinity Purification of Nucleus- or Cytoplasm-Localized lncRNA-Protein Complexes Formed In Vivo.

With recent emergence of huge number of long noncoding RNAs (lncRNAs), purification of lncRNA-protein (lncRNP) complexes is fundamental to understand the role of lncRNA and its biological function. However, lncRNP purification is still a daunting challenge. Here we describe a protocol to purify lncRNP formed in vivo with MS2-MBP-based affinity purification. Inducible lncRNA tagged with MS2 RNA hairpins is introduced into cells of interest, and RNP on tagged lncRNA is formed in vivo. MS2-MBP fusion protein is expressed in Escherichia coli and purified with amylose resin and HiTrap heparin column. The MS2 part of MS2-MBP fusion protein binds to the hairpins, and MBP part binds to amylose resin. We also describe a protocol to separate the nucleus and the cytoplasm so that lncRNP localized in the nucleus or cytoplasm can be individually purified. The amount of lncRNP purified is well sufficient for mass spectrometry analysis.