Identification And Characterization Of A Virus Inducible Non Coding RNA (VINC)

Abstract

Non-protein coding eukaryotic genome sequences often referred to as junk DNA are estimated to encode several non-coding RNAs (ncRNAs) which may account for nearly 98% of all genomic output in humans. The output of such a wide spread transcription in eukaryotes consists of intronic, antisense and small RNAs. In addition to the classical ncRNAs such as rRNA, tRNA and small nucleolar RNAs, the eukaryotic genome encodes two distinct categories of ncRNAs, referred to as small ncRNAs and long mRNA–like ncRNAs (mlncRNAs). The long ncRNAs, which are transcribed by RNA Polymerase II, spliced and polyadenylated, are implicated in a number of regulatory processes such as imprinting, X-chromosome inactivation, DNA demethylation, transcription, RNA interference, chromatin structure dynamics and antisense mediated regulation. Expression of noncoding RNAs is altered during stress conditions and a large number of such transcripts have been identified of late. This study has identified a novel ncRNA whose expression is upregulated during viral infection of mouse brain. While we have named this RNA as VINC or virus inducible ncRNA, others have named it as NEAT1 (Hutchinson et al., 2007) and Men (Sunwoo et al., 2008). VINC/NEAT1/Men is associated with a distinct nuclear domain called paraspeckles Using a cell line as well as an animal model system we have investigated VINC in great detail and based on these studies we report that VINC is a nuclear ncRNA that localizes to paraspeckles and it interacts with the paraspeckle protein, P54nrb in both cell line model system as well as in animal tissues by a combination of in vitro and in vivo methods. We have also mapped the domains within VINC that are involved in P54nrb interactions. Till date, the only other RNA known to localise to paraspeckles is CTN-RNA. While CTN-RNA is a protein coding RNA, VINC does not code for a protein and thus VINC is the first ncRNA to be localized to paraspeckles. Further, the mechanism of nuclear retention of these two paraspeckle RNAs appears to be distinct. In case of CTN-RNA, it has been clearly shown that it is A-I edited and such hyperedited RNAs are retained by the p54/nrb mediated complex in nucleus (Zhang and Carmichael, 2001). However the mechanism by which VINC is retained in nucleus is not clear. There is apparently no A-I editing in VINC and hence VINC retention in the nucleus by binding to nuclear proteins such as p54/nrb might involve a different mechanism. It is well established of late that nuclear matrix retains RNAs and that there is a population of poly (A) RNA that is retained in nucleus (Huang et al.,1994 ; Carter et al.,1991). However the significance of such retention is not clear but it is believed that it might be important for some constitutive functions in nucleus (Nickerson et al., 1989). More investigations are needed to understand the exact functions of nuclear RNAs such as VINC in supporting the nuclear architecture. P54nrb is a multi functional nuclear protein that mediates most of its functions in association with PSF (Shav-Tal and Zipori, 2002). Phosphorylation status of P54nrb is a key determinant for its localisation to various nuclear regions. P54nrb is a multiphosphorylated protein during mitosis and its phosphorylation is mediated by PIN-1 at its C-terminus (Proteau et al., 2005). Tyrosine phosphorylation of P54nrb is essential for it to be retained in nuclear matrix (Otto et al., 2001). The N-terminal phosphorylation is speculated but not much has been investigated. The protein has two distinct RNA recognition motifs (RRMs) in its N-terminus that are responsible for its RNA binding activity. The significance of the p54/nrb-PSF heterodimer cannot be undermined as they have been shown to be important during HIV replication. The dimer is recruited by viral machinery and P54nrb has been shown to be exported to cytosol for binding to replicative complexes (Zolotukhin et al., 2003). During adenoviral replication in nucleus many SR proteins are recruited to viral replication foci and rearrangement of speckle components happen. It has been shown with respect to speckles that nuclear domains are highly dynamic and exchange of proteins depends upon the transcriptional status of cell (Lamond and Spector, 2003). Flaviviral replication complexes are hosted in nucleus and ~20% of this complex docks in nucleus and serves as an alternate site for viral replication. The presence of viral replicative complexes alters the nuclear organisation and hence modulation of gene expression is expected (Uchil et al., 2006). The up regulation of nuclear ncRNA such as VINC is definitively one of those events associated with viral replication and definitively one needs to study the various changes carefully to understand the role of VINC in virus life cycle and/or viral pathogenesis. VINC interaction with the multi-functional nuclear protein P54nrb raises interesting aspects related to function of P54nrb in JEV infection. Knockdown of P54nrb in human myeloid cell line results in abnormal size of paraspeckles and impairs chondrogenesis (Hata et al., 2008). PSF-P54nrb complex can divert many of HIV gag RNA complexes to paraspeckles thus trying to restrict viral replication. However the exact relationship between paraspeckles and its constituent proteins is not clear. The presence of ncRNA adds another new dimension to paraspeckles. It is unclear whether the ncRNA VINC is essential for paraspeckle structure but a recent study indicates that Men (VINC/NEATI) RNA may be essential for paraspeckle formation (Sunwoo et al., 2008). The exact function VINC in neuronal as well as non-neuronal cell nuclei remains elusive and more investigations are need to understand these aspects.