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J Gen Virol 87 (2006), 1991-1995; DOI 10.1099/vir.0.81768-0

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© 2006 Society for General Microbiology

Short Communication

Identification and characterization of a virus-inducible non-coding RNA in mouse brain

Sougata Saha{dagger}, Sreenivasa Murthy{dagger} and Pundi N. Rangarajan

Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

Correspondence
Pundi N. Rangarajan
pnr{at}biochem.iisc.ernet.in


   ABSTRACT
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Infection of mice with Japanese encephalitis virus or Rabies virus results in the activation of a gene encoding a novel, non-coding RNA (ncRNA) in the mouse central nervous system. This transcript, named virus-inducible ncRNA (VINC), is identical to a 3.18 kb transcript expressed in mouse neonate skin (GenBank accession no. AK028745 [GenBank] ) that, together with a number of unannotated cDNAs and expressed sequence tags, is grouped in the mouse unigene cluster Mm281895. VINC is expressed constitutively in early mouse embryo and several adult non-neuronal mouse tissues, as well as a murine renal adenocarcinoma (RAG) cell line. Northern blotting of nuclear and cytoplasmic RNAs revealed that VINC is localized primarily in the nucleus of RAG cells and is thus a novel member of the nuclear ncRNA family.

A schematic representation of the AK028745 cDNA is available as supplementary material in JGV Online.

{dagger}These authors contributed equally to this work. Back


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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 (http://research.imb.uq.edu.au/rnadb). 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. 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 regulation. In addition, long mRNA-like ncRNAs such as MALAT-1, BC-1 and BC-200 serve as prognostic markers for cancer, whilst the prion-associated RNAs LIT-1, SCA-8 etc. are implicated in a number of neurological disorders (Costa, 2005Down). Thus, identification and characterization of novel ncRNAs and constant updating of the mammalian RNome are essential for the complete deciphering of genome biology and understanding mammalian gene regulation.

Infection of vertebrate cells by viruses results in dramatic changes in the host transcriptome and these changes can be either detrimental or beneficial to the virus. Availability of mammalian genome-sequence data and microarray technologies have led to the discovery of common host genes involved in the antiviral response and unique host genes that can serve as ‘biomarkers' of a specific virus infection, as well as several unannotated, virus-inducible host genes of unknown function (Jenner & Young, 2005Down). By using the subtraction-hybridization technique, we previously identified several Japanese encephalitis virus (JEV)- and Rabies virus-inducible mouse central nervous system (CNS) genes (Saha & Rangarajan, 2003Down). Further screening of the subtracted cDNA library led to the identification of many more JEV-inducible genes (S. Saha & P. N. Rangarajan, unpublished data) and one of them, designated clone #150, had >99 % identity to a mouse neonate skin cDNA (GenBank accession no. AK028745 [GenBank] ) as well as several murine cDNAs expressed in neuronal and non-neuronal tissues and mammary tumours, all of which are annotated as transcripts of unknown function (Fig. 1aDown). A detailed study was therefore undertaken to characterize this gene further. RNA was isolated from normal or JEV-infected outbred Swiss mice (Saha & Rangarajan, 2003Down) and Northern blot analysis was carried out with a radiolabelled clone #150 cDNA probe. An approximately 3.1 kb transcript was detectable in JEV-infected, but not in normal, mouse brain in Northern blots (Fig. 1bDown), which matched the size of a mouse neonate skin cDNA clone (3180 bp) described in GenBank (accession no. AK028745 [GenBank] ). A series of experiments confirmed that the JEV-inducible RNA in mouse brain is the same as that encoded by the AK028745 [GenBank] cDNA, as indicated below. The regions corresponding to bp 801–1730 (probe A) and bp 2358–3169 (probe B) of the AK028745 [GenBank] cDNA could be amplified from JEV-infected mouse brain RNA by RT-PCR using primer pairs 5'-CTCACTCTGAGGTTAAGGGG-3' and 5'-TAACTTGCGCCTTCCCACTG-3' (for probe A) and 5'-CCCTGACCTAGGCAGGCCAC-3' and 5'-CTCAAACCTTTATTTTGCTGTAAAGGG-3' (for probe B), designed based on the nucleotide sequence of GenBank accession no. AK028745 [GenBank] . These PCR products, when used as radiolabelled probes, hybridized to the same mRNA species in the JEV-infected mouse brain as did the clone #150 cDNA (Fig. 1bDown). We also generated digoxigenin (DIG)-labelled strand-specific riboprobes based on the nucleotide sequence with GenBank no. AK028745 [GenBank] and the results presented in Fig. 1(c)Down indicate that only the antisense riboprobe hybridizes to an approximately 3.1 kb transcript in Northern blots of JEV-infected mouse brain RNA. Further, an approximately 3.1 kb RT-PCR product could be obtained from JEV-infected mouse-brain RNA (Fig. 1dDown, lane 2) by using primer pair 5'-GAGGAGTTAGTGACAAGGAGGGC-3' and 5'-CTCAAACCTTTATTTTGCTGTAAAGGG-3', corresponding to the 5' and 3' ends of the AK028745 [GenBank] cDNA, and the nucleotide sequence of the PCR product was identical to the reported sequence of the AK028745 [GenBank] cDNA (data not shown). Based on these results, we concluded that the 3180 bp mouse neonate skin mRNA represented by GenBank accession no. AK028745 [GenBank] and the JEV-inducible mouse-brain transcript hybridizing to clone #150 described in this study are identical.


Figure 1
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  		Fig. 1. Establishing the identity of the clone #150 cDNA and analysis of its expression in mouse brain during rabies virus infection. (a) Clone #150 and its homologues as identified by a BLAST search of GenBank. The thin line represents a region that is spliced out as an intron in BC025215 cDNA. (b) Northern blot of RNA isolated from brain of normal mice (lane 1) or JEV-infected mice (lane 2), probed sequentially with clone #150 cDNA, probes A or B and finally with GAPDH cDNA. (c) Northern blots of JEV-infected mouse-brain RNA were probed with DIG-labelled sense (S) or antisense (As) clone #150 riboprobes. (d) A 3.2 kb cDNA (lane 2) could be obtained by RT-PCR from JEV-infected mouse-brain RNA by using primers corresponding to the 5' and 3' ends of the AK028745 cDNA. Lane 1, DNA molecular mass markers (in kb). (e) Northern analysis of Rabies virus-infected mouse-brain RNA using radiolabelled clone #150 cDNA (i). Brain RNA was isolated from uninfected mice (lane 1), mice inoculated with Rabies virus via the intracerebral route and sacrificed at days 3 (lane 2) and 6 (lane 3) post-inoculation or mice inoculated with Rabies virus in footpads and sacrificed at days 3 (lane 4) and 6 (lane 5) post-inoculation. The blot was later reprobed with a GAPDH probe (ii). Arrows indicate the ~3.18 kb AK028745 transcript.

 
As the AK028745 [GenBank] transcript has not been reported to be induced by any virus except JEV, as described in this study, we examined its expression during rabies virus infection by using the well-characterized murine intracerebral and peripheral rabies virus challenge models (Biswas et al., 2001Down; Saha & Rangarajan, 2003Down). The results indicate that inoculation of Rabies virus through the intracerebral route (Fig. 1eUp, lanes 2 and 3) or in the footpads (Fig. 1eUp, lanes 4 and 5) leads to induction of expression of the AK028745 [GenBank] transcript in mouse brain.

Analysis of the AK028745 [GenBank] cDNA sequence for an open reading frame (ORF), using an ORF search tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), indicated that the mRNA is unlikely to encode a protein, as no significant ORFs could be detected in the GenBank AK028745 [GenBank] nucleotide sequence (see Supplementary Fig. S1, available in JGV Online). Also, no major ORFs are detectable in the other murine cDNAs listed in Fig. 1(a)Up (data not shown). Thus, the AK028745 [GenBank] cDNA and other related cDNAs appear to be non-coding RNAs (ncRNAs) and, in view of the upregulation of the former in mouse brain during virus infection, it was named virus-inducible ncRNA (VINC).

As several transcripts identical to VINC have been reported from non-neuronal tissues and mammary tumours (Fig. 1aUp), we examined VINC expression in multiple mouse tissues by RNA dot-blot analysis using a Mouse RNA Master Blot kit (catalogue no. 7770-1; Clontech). The results demonstrate that VINC is expressed constitutively at high levels in several adult tissues, such as liver, lung, kidney, heart and skeletal muscle, as well as in 7-day-old mouse embryo, but not in late embryonic stages, adult brain or testis (Fig. 2aDown). These results were further confirmed by Northern blot analysis of selected mouse tissues. A transcript similar in size to that induced in JEV-infected mouse brain is expressed at high levels in heart, kidney, liver and muscle, but not brain or testis (Fig. 2bDown).


Figure 2
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  		Fig. 2. Analysis of VINC expression in various mouse tissues. (a) Mouse multiple-tissue RNA dot blot. (b) Northern blots. The blots were probed with radiolabelled clone #150 cDNA. Arrow indicates the ~3.18 kb VINC transcript.

 
The constitutive expression of VINC in a number of non-neuronal tissues, including kidney, led us to examine its expression in the RAG mouse renal adenocarcinoma cell line. The data presented in Fig. 3Down(a) indicate that VINC is expressed constitutively in this cell line. We also examined the intracellular localization of VINC in RAG cells by Northern blot analysis of total cellular RNA, as well as RNA isolated from nucleus and cytoplasm (PARIS kit; Ambion). The results presented in Fig. 3(b)Down indicate that VINC is detectable only in the nucleus, but not the cytoplasm, under these conditions, whereas GAPDH is localized predominantly in the cytosol and mouse U1 RNA (Kato & Harada, 1985Down) is localized primarily in the nucleus. The nuclear localization of VINC was further confirmed by RNA in situ hybridization (Décimo et al., 1995Down) in RAG cells by using DIG-labelled sense and antisense riboprobes. In the case of the VINC antisense riboprobe, the staining was detectable predominantly in the nucleus, as opposed to the cytoplasmic staining of GAPDH (Fig. 3cDown).


Figure 3
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  		Fig. 3. Analysis of VINC expression in the RAG murine renal adenocarcinoma cell line. (a) Demonstration of VINC (arrow) expression in RAG cells by Northern analysis of RAG total RNA using a radiolabelled clone #150 cDNA probe. (b) Northern blot analysis of total (T), cytosolic (C) and nuclear (N) RNA isolated from RAG cells. The blot was probed sequentially with radiolabelled clone #150 cDNA, GAPDH cDNA or a 5' end-labelled U1 RNA oligonucleotide probe. The mouse U1 RNA probe was prepared by 5'-end labelling of an oligonucleotide (5'-AATGGATAAGCCTCGCCCTGGGAAAACCACCTTCGTGATCATGGTATCTCCCCTGCAGGT-3') corresponding to a region between bp 8 and 68 of mouse U1a-1 RNA (Kato & Harada, 1985Down). (c) RNA in situ hybridization was carried out in RAG renal adenocarcinoma cells, using DIG-labelled sense and antisense riboprobes specific for VINC and GAPDH.

 
Global changes in host-gene expression have been reported for more than 26 different species of bacteria, 30 different types of virus, several pathogenic yeasts and parasites (Jenner & Young, 2005Down). Such studies have led not only to the discovery of new functions for well-characterized genes and/or proteins, but also identification of novel genes that were not known to play a role in host response to viral infection. However, information on global gene-expression changes in the mouse CNS during infection by neurotropic viruses and other agents is just beginning to emerge and is currently limited to Rabies virus (Prosniak et al., 2001Down), Sindbis virus (Johnston et al., 2001Down; Labrada et al., 2002Down), scrapie agent (Booth et al., 2004Down), coronavirus (Gruslin et al., 2005Down) and JEV (Saha & Rangarajan, 2003Down). Using JEV and Rabies virus as model pathogens, we have been examining gene-expression changes induced by neurotropic viruses in the mouse CNS (Saha & Rangarajan, 2003Down). We identified a number of JEV-inducible protein-coding genes (S. Saha & P. N. Rangarajan, unpublished data) that have already been shown to have established roles in the host response to viral infection. We therefore focused our attention on a novel, unannotated transcript, referred to in this study as clone #150, that has not been studied so far. Analysis of sequences in GenBank, as well as a series of experiments (Fig. 1a–dUp), confirmed that the clone #150 cDNA is identical to a 3180 bp 10-day-old mouse neonate skin cDNA (GenBank accession no. AK028745 [GenBank] ) that, together with a number of related cDNAs and expressed sequence tags, constitutes the mouse unigene cluster Mm281895 ( http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm&CID=281895 ). Interestingly, no major ORFs could be discerned in any of these cDNAs (see Supplementary Fig. S1, available in JGV Online). Thus, GenBank no. AK028745 [GenBank] and other cDNAs of the Mm281895 mouse unigene cluster appear to be ncRNAs. Further, the AK028745 [GenBank] transcript is induced in brain not only by JEV, but also by Rabies virus (Fig. 1eUp). In view of its activation in mouse brain by two different neurotropic viruses, the AK028745 [GenBank] transcript has been named virus-inducible ncRNA or VINC.

Our studies demonstrate that VINC is expressed constitutively in a number of non-neuronal tissues, as well as during early embryonic development (Fig. 2Up). These results are comparable to the expression profile of AK028745 [GenBank] and other cDNAs of the Mm281895 unigene cluster reported in computational databases (http://symatlas.gnf.org/SymAtlas/, http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.281895). Constitutive expression of VINC in adult mouse kidney led us to examine its expression in the RAG mouse renal adenocarcinoma cell line. The results indicate that not only is VINC expressed constitutively in this cell line, but the transcript is detectable only in the nuclear and not the cytosolic RNA (Fig. 3Up). Thus, VINC is a novel member of the nuclear ncRNA family, which includes transcripts such as Xist (Brockdorff et al., 1992Down), TUG-1 (Young et al., 2005Down), EVF-1 (Kohtz & Fishell, 2004Down), Ks-1 (Sawata et al., 2002Down), NTT (Liu et al., 1997Down), Khps-1 (Imamura et al., 2004Down), PAT-1 (Chao et al., 1998Down) and rOX-1 and -2 (Franke & Baker, 1999Down).

Although ncRNAs are shown to carry out a number of functions, the cellular roles of several ncRNAs still remain unknown (Storz, 2002Down). The first step towards understanding the function(s) of ncRNAs is to study their spatial- and temporal-expression patterns and develop a suitable experimental system for carrying out functional studies. Thus, the identification of VINC in mouse brain and demonstration of its nuclear localization in a cell line should pave the way for further functional characterization of this novel ncRNA.


   ACKNOWLEDGEMENTS
 
This study was supported by the Swarnajayanthi fellowship of the Department of Science and Technology, New Delhi, India, awarded to P. N. R.


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Received 16 December 2005; accepted 2 March 2006.


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