Background

The mammalian cell nucleus is highly organized and composed of multiple distinct substructures called nuclear bodies (NBs). So far ~15 NBs have been identified as subnuclear membraneless granular structures containing various proteins and RNA factors, many of which function as sites of biogenesis, maturation, storage, and sequestration of specific RNAs, proteins, and/or ribonucleoprotein (RNP) complexes (Mao et al., 2011; Sleeman and Trinkle-Mulcahy, 2014) (Table 1).

Table 1. Nuclear bodies in mammalian cells


Some NBs are constructed on specific long noncoding RNAs (lncRNAs) called architectural RNAs (arcRNAs), which are defined as structural core of NBs (Chujo et al., 2016). The arcRNA-dependent NBs are composed of numerous RNA-binding proteins that interact with the arcRNAs. The most remarkable example is the paraspeckle, which is composed of several characteristic RNA-binding proteins (Fox et al., 2002; Prasanth et al., 2005). RNase treatment disintegrates the paraspeckle structure (Fox et al., 2005). Nuclear paraspeckle assembly transcript 1 (NEAT1), a lncRNA, localizes exclusively to paraspeckles and acts as an arcRNA of these massive RNP complexes (Chen and Carmichael, 2009; Clemson et al., 2009; Sasaki et al., 2009; Sunwoo et al., 2009).

Presently, two lncRNAs are classified as arcRNAs in addition to NEAT1, namely, intergenic spacer lncRNAs for nucleolar detention center (Audas et al., 2012) and human satellite III lncRNA for nuclear stress body (Biamonti and Vourc’h, 2010) (Table 1). It is expected that additional arcRNAs remain to be characterized in mammalian cells. Here, we describe a novel method called ‘RNase sensitivity screening’ to identify novel arcRNA-dependent NBs by screening for nuclear foci whose structures are disintegrated by RNase treatment. This method employed a Venus-tagged human full-length (FLJ) cDNA library (32,651 clones), which was originally constructed during the NEDO full-length human cDNA sequencing project in Japan (FLJ-PJ), and obtained 571 cDNA clones whose products (463 proteins) localize in certain nuclear foci (Hirose and Goshima, 2015; Naganuma et al., 2012). We explored whether the respective nuclear focus was abolished or diffused upon RNase treatment after cell permeabilization to select candidate RNase-sensitive nuclear foci that potentially contain arcRNAs (Figure 1). We identified 32 Venus-tagged proteins that required RNA for their localization in distinct nuclear foci (Mannen et al., 2016). Immunostaining of the corresponding endogenous proteins confirmed that the Sam68 nuclear body (SNB) was an RNase-sensitive NB. In the following protocol, we describe the detailed procedure of the RNase sensitivity screening. This protocol is for screening for RNase-sensitive NBs under normal conditions in HeLa cells, but it should be applicable to other cell lines under various conditions.


Figure 1. Outline of RNase sensitivity screening of NBs. Venus-tagged human FLJ cDNA clones were transfected into HeLa cells. cDNA clones whose products localized to certain nuclear foci were selected (571 clones). Subsequently, the RNase sensitivity of the nuclear foci labeled by Venus was investigated (32 clones). To this end, the cells were permeabilized with 2% Tween 20, followed by treatment with an RNase mixture. Bar = 10 μm.