Dual Short Upstream Open Reading Frames Control Translation of a Herpesviral Polycistronic mRNA

Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America.
PLoS Pathogens (Impact Factor: 7.56). 01/2013; 9(1):e1003156. DOI: 10.1371/journal.ppat.1003156
Source: PubMed


Author Summary
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of multicentric Castleman's disease, primary effusion lymphoma and Kaposi's sarcoma. KSHV expresses a number of transcripts with the potential to generate multiple proteins, yet relies on the cellular translation machinery that is primed to synthesize only one protein per mRNA. Here we report that the viral transcript encompassing ORF35–37 is able to direct synthesis of two proteins and that the translational switch is regulated by two short upstream open reading frames (uORFs) in the native 5′ untranslated region. uORFs are elements commonly found upstream of mammalian genes that function to interfere with unrestrained ribosomal scanning and thus repress translation of the major ORF. The sequence of the viral uORF appears unimportant, and instead functions to position the translation machinery in a location that favors translation of the downstream major ORF, via a reinitiation mechanism. Thus, KSHV uses a host strategy generally reserved to repress translation to instead allow for the expression of an internal gene.

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    • "Secondly, to further validate our findings that internal translation is dependent on a functional cap, ribosomal scanning or GJA1-43k translation, we inserted a highly stable hairpin (HP7) into the 5′UTR (ΔG = −61 kcal/mol; see Additional file 1: Methods and Additional file 2: Figure S1), previously described to efficiently prevent ribosomal scanning and cap-dependent translation [21,22]. Additionally, to generate a control readout of cap-independent translation within the same transcript, we inserted (downstream of Cx43) both the encephalomyocarditis virus (EMCV) IRES sequence plus an enhanced green fluorescent protein (EGFP) reporter. "
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    ABSTRACT: Background Connexin 43 (Cx43), the most widely expressed gap junction protein, is associated with a number of physiological and pathological conditions. Many functions of Cx43 have been shown to be independent of gap junction formation and only require the expression of Cx43 C-terminal fragments. Recent evidence demonstrated that naturally occurring C-terminal isoforms can be generated via internal translation. Findings Here, we confirm that C-terminal domains of Cx43, particularly the major 20-kDa isoform, can be independently generated and regulated by internal translation of the same single GJA1 gene transcript that encodes full-length Cx43. Through direct RNA transfection experiments, we provide evidence that internal translation is not due to a bona fide cap-independent IRES-mediated mechanism, as upstream ribosomal scanning or translation is required. In addition to the mTOR pathway, we show for the first time, using both inhibitors and cells from knockout mice, that the Mnk1/2 pathway regulates the translation of the main 20-kDa isoform. Conclusions Internal translation of the Cx43 transcript occurs but is not cap-independent and requires translation upstream of the internal start codon. In addition to the PI3K/AKT/mTOR pathway, the major 20-kDa isoform is regulated by the Mnk1/2 pathway. Our results have major implications for past and future studies describing gap junction-independent functions of Cx43 in cancer and other pathological conditions. This study provides further clues to the signalling pathways that regulate internal mRNA translation, an emerging mechanism that allows for increased protein diversity and functional complexity from a single mRNA transcript.
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    ABSTRACT: Technological advances in genome-wide transcript analysis, referred to as the transcriptome, using microarrays and deep RNA sequencing methodologies are rapidly extending our understanding of the genetic content of the gammaherpesviruses (γHVs). These vast transcript analyses continue to uncover the complexity of coding transcripts due to alternative splicing, translation initiation and termination, as well as regulatory RNAs of the γHVs. A full assessment of the transcriptome requires that our analysis be extended to the virion and exosomes of infected cells since viral and host mRNAs, miRNAs, and other noncoding RNAs seem purposefully incorporated to exert function upon delivery to naïve cells. Understanding the regulation, biogenesis and function of the recently discovered transcripts will extend beyond pathogenesis and oncogenic events to offer key insights for basic RNA processes of the cell.
    No preview · Article · May 2013
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    ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes over 90 genes and 25 microRNAs (miRNAs). KSHV lifecycle is tightly regulated to ensure persistent infection in the host. Particularly, miRNAs, which primarily exert their effects by binding to the 3' untranslated regions (3' UTRs) of target transcripts, have recently emerged as key regulators of KSHV lifecycle. While works with RNA crosslinking immunoprecipitation approach have identified numerous targets of KSHV miRNAs, few are of viral origins because most KSHV 3' UTRs have not been characterized. Thus, the extents of viral genes targeted by KSHV miRNAs remain elusive. Here, we report the mapping of the 3' UTRs of 74 KSHV genes and effects of KSHV miRNAs on the control of these 3' UTR-mediated gene expressions. This analysis reveals new bicistronic and polycistronic transcripts of KSHV genes. Due to the 5' -distal open reading frames (ORFs), KSHV bicistronic or polycistronic transcripts have significantly longer 3' UTRs than its monocistronic transcripts. Furthermore, screening of the 3' UTR reporters has identified 28 potential new targets of KSHV miRNAs, of which 11 (39%) are bicistronic or polycistronic transcripts. Reporter mutagenesis demonstrates that miR-K3 specifically targets ORF31/32/33 transcripts at the lytic locus via two binding sites in the ORF33 coding region, while the miR-K10a-3p, miR-K10b-3p and their variants target ORF71/72/73 transcripts at the latent locus through distinct binding sites in both 5' -distal ORFs and intergenic regions. Our results indicate that KSHV miRNAs frequently target the 5' -distal coding regions of bicistronic or polycistronic transcripts and highlights the unique features of KSHV miRNAs in regulating its gene expression and lifecycle.
    Full-text · Article · Oct 2013 · Journal of Virology
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