What’s behind the dot? - Holistic Content Analysis of the Paraspeckle Complex -

In the nucleus of higher eucaryotes one can distinguish between different substructures with varied roles in genome maintenance and gene regulation. Discovered in 2002, Paraspeckles are one of these nuclear bodies. They are visible under the microscope as little dots in the range of 0.5-1 µm with varying numbers per cell (Fox et al., 2002). Paraspeckles are involved in gene regulation by trapping adenosine to inosine (A to I) hyperedited RNA within the nucleus which may be a widely used mechanism within a variety of different cellular contexts (Zhang and Carmichael, 2001, Chen and Carmichael, 2009). Currently, there are three proteins known to build up the core of the paraspeckle, namely PSPC1, NONO and SFPQ (Fox et al., 2002, Prasanth et al., 2005). Additionally the long non-coding RNA NEAT1 plays a crucial role for the structural integrity of Paraspeckles. The two isoforms of NEAT1 (NEAT1_v1, 3.7 kb and NEAT1_v2, 23 kb) seem to build up the scaffold to which the core Paraspeckle proteins are recruited to form the speckle by not only protein-protein but also protein-RNA interactions (Clemson et al., 2009, Sasaki et al., 2009, Sunwoo et al., 2009 and Souquere et al., 2010). We are investigating the full protein- and RNAome of this mega complex as this will be the key to further understanding of the paraspeckular mechanisms and the underlying functions. We use protein- and RNA- affinity techniques (Hogg and Collins, 2007) to purify Paraspeckle material from HeLa cells. This will be subject to Mass Spectrometry and Deep Sequencing. Here I describe the generation and the characterisation of a new cell line stably over expressing a tagged, non-coding, Paraspeckle specific RNA.