Nuclear-BRITE: an enhanced MS2 system for live-cell imaging of nuclear RNAs

Analysing the dynamic behaviour of specific RNAs using MS2 stem-loop arrays bound by fluorescently tagged proteins, provides important insights into their biological function. However, high levels of background resulting from the accumulation of unbound stem-loop binding proteins presents a significant limitation to this approach, especially in relation to RNAs that function in the cell nucleus. Here we describe Nuclear-BRITE, an improved MS2-based methodology that significantly reduces nuclear background signal without perturbing the abundance, localisation or function of RNAs of interest. We demonstrate the applicability of this novel approach for fast live-cell single-molecule imaging of endogenously tagged nuclear long non-coding RNAs (Xist, Kcnq1ot1, Malat1), processed mRNAs (Polr2a mRNA), mRNA introns (Hprt and Hspg2), and short functional RNAs (7SK, U1 snRNA, U3 snoRNA, 5S rRNA and scaRNA-8). Accordingly, Nuclear-BRITE provides a valuable new tool to enable live-cell imaging-based analysis of the full range of RNAs that function in the cell nucleus.