Epstein–Barr Virus-Induced Expression of a Novel Human Vault RNA

Abstract

Non-protein-coding RNAs (ncRNAs) have recently emerged on the scene of genomic research as prominent players in the regulation of gene expression. Many functionally characterized ncRNAs have been shown to be differentially expressed in various organisms during specific environmental or developmental conditions, thus establishing regulatory networks crucial for shaping cellular life. Here, we show that the expression of vault RNAs (vtRNAs) is specifically up-regulated in human lymphocytes upon infection by γ-herpesviruses, such as the Epstein–Barr virus and Kaposi's sarcoma virus. vtRNAs are ncRNAs that are integral to the vault complex, a gigantic (13 MDa) hollow ribonucleoprotein particle with a thus far elusive biological role. Stimulation of vtRNA expression by the Epstein–Barr virus was evident for all three canonical vtRNAs (hvg1–hvg3) and also for a novel ncRNA candidate, initially termed CBL-3. This ncRNA shares clear primary- and secondary-structure similarities with the three known vtRNAs. Importantly, CBL-3 co-sediments with intact vault particles in density gradients of various human cell lines, thus strongly indicating this ncRNA as a novel, fourth vault-complex-associated RNA.

Introduction

In the recent past, the importance of the surprisingly diverse class of non-protein-coding RNA (ncRNA) molecules has been widely recognized. All transcriptomes analyzed to date consist of two distinct classes of RNA molecules: the mRNAs that are subject to translation into proteins by the ribosome and ncRNAs that exert their cellular functions on the level of RNA.¹ ncRNAs play essential and prominent roles in many cellular processes, in particular in the regulation of gene expression. The physicochemical characteristics of RNA allow ncRNAs to fulfill a diverse range of cellular roles through various mechanisms.² The functions of ncRNAs can be directly catalytic, as in the case of ribozymes, or indirect, for example, by base-pairing to a target RNA/DNA and thereby conferring specificity to (an) associated protein(s).³ ncRNAs are present in all three domains of life, that is, Archaea, Bacteria, and Eukarya, while their number rises as the complexity of the organism increases. Bacteria and more primitive single-cell eukaryotes encode only few ncRNA genes, and the genomes of multicellular organisms and especially mammals have been predicted to code for up to several 100,000 ncRNAs.⁴ Some estimations even suggest that around 97–98% of the transcriptional output of the human genome is ncRNA.⁵ Since the protein-coding gene content does not dramatically differ between bacterial and mammalian genomes, it was suggested that ncRNAs contribute to the complex networks needed to regulate cell function, implying that RNA has evolved a new significance in the genetic programming of higher organisms.

ncRNA genes are not restricted to the nuclear genome of a cell but have also been found to be encoded in the mitochondria and chloroplast genomes and recently even in viral genomes. In human disease caused by viruses, host cell infection is a tightly controlled process with regulated temporal expression of viral as well as cellular genes. Recently, it has been shown that virus infection indeed changes the RNA expression profile also on the level of ncRNAs. In 2004, first reports were presented, demonstrating that expression of virus-derived ncRNAs can regulate or modulate virulence by inhibiting the defense mechanisms of the host cell such as the small interfering RNA machinery⁶ or by targeting viral DNA polymerase.⁷ Today, more than 120 viral microRNAs (miRNAs) as well as several longer virus-derived ncRNAs (> ~ 100 nucleotides) have been identified (reviewed in Ref. 8). Additionally, host-encoded ncRNAs have been characterized, which are expressed in response to the virus infection to regulate virus-relevant processes such as apoptosis, immune response, and tumorigenesis.

Most virus-derived ncRNAs have been found in the large DNA genome herpesvirus family, such as the Epstein–Barr virus (EBV). EBV, a lymphotropic virus of the γ-herpesviridae family with a linear double-stranded genome of ~ 172 kb, is associated with a heterogeneous group of human tumors including Burkitt's lymphoma.⁹ EBV encodes at least 23 miRNAs¹⁰ and two longer ncRNAs, designated as EBV-encoded RNA (EBER) 1 and EBER 2, transcripts of 167 and 172 nucleotides length, respectively.¹¹ In a recent genomic screen using a novel setup of the subtractive hybridization approach (SHORT, or subtractive hybridization of non-coding RNA transcripts), we identified several ncRNAs whose expression was found to be significantly up-regulated in the presence of EBV.¹² In this study, 21 ncRNAs species, including the host-encoded miRNAs miR-21, miR-155, or miR-146a,¹³ as well as five novel ncRNA candidates, have been shown to be overexpressed. The ncRNAs that were most strongly up-regulated in the presence of EBV were the three host-encoded vault RNAs (vtRNAs).¹² In the primary human cord blood lymphocyte (CBL) cell line that was established by transformation with EBV, vtRNAs were up-regulated 20- to ~ 1200-fold.¹² Surprisingly, one of the five newly identified ncRNA candidates showed primary- and secondary-structure similarities with the three canonical vtRNAs and was referred to as CBL-3 (for CBL-derived ncRNA)¹² (Fig. 1a). Thus, this transcript might represent a novel member of the vtRNA family. In the human genome, the genes hvg1–hvg3 encoding the three known human vtRNAs 1–3 (99, 88, and 89 nucleotides in size, respectively) are located in a cluster on chromosome 5 close to the PCDHA (protocadherin-α) genes (P. Stadler, personal communication)‡. In response to the work of Stadler (personal communication)‡ and the present study, the HUGO Gene Nomenclature Committee§ will, in its next release, revise the nomenclature of vtRNA genes, and accordingly, these transcripts should be referred to as vtRNA1-1, vtRNA1-2, and vtRNA1-3. The vtRNAs are integral to the so-called vault complex, a large (13 MDa), hollow, barrel-shaped ribonucleoprotein (RNP) complex.¹⁵ The vault RNP consists of multiple copies of three different highly conserved proteins [major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein 1 (TEP1)] and at least six copies of vtRNAs.¹⁶ Very little is known about the function of this ncRNA class and the entire vault complex. The vault complex has been implicated in various tasks such as multidrug resistance, transport, signaling (see Ref. 15 and references therein), apoptosis resistance,¹⁷ or innate immunity;¹⁸ however, its biological function has yet to be identified.