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: 8.06). 01/2013; 9(1):e1003156. DOI: 10.1371/journal.ppat.1003156
Source: PubMed

ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) protein kinase, encoded by ORF36, functions to phosphorylate cellular and viral targets important in the KSHV lifecycle and to activate the anti-viral prodrug ganciclovir. Unlike the vast majority of mapped KSHV genes, no viral transcript has been identified with ORF36 positioned as the 5'-proximal gene. Here we report that ORF36 is robustly translated as a downstream cistron from the ORF35-37 polycistronic transcript in a cap-dependent manner. We identified two short, upstream open reading frames (uORFs) within the 5' UTR of the polycistronic mRNA. While both uORFs function as negative regulators of ORF35, unexpectedly, the second allows for the translation of the downstream ORF36 gene by a termination-reinitiation mechanism. Positional conservation of uORFs within a number of related viruses suggests that this may be a common γ-herpesviral adaptation of a host translational regulatory mechanism.

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    ABSTRACT: The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF36 protein kinase is translated as a downstream gene from the ORF35-37 polycistronic mRNA via a unique mechanism involving short upstream open reading frames (uORFs) located in the 5' untranslated region. Here, we confirm that ORF35-37 is functionally dicistronic during infection and demonstrate that mutation of the dominant uORF restricts KSHV replication. Leaky scanning past the uORFs facilitates ORF35 expression, while a reinitiation mechanism after the uORFs enables ORF36 translation.
    Journal of Virology 03/2014; · 4.65 Impact Factor
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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.
    Cell Communication and Signaling 01/2014; 12:31. · 4.67 Impact Factor
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    ABSTRACT: Successful viral infection entails a choreographic regulation of viral gene expression program. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes numerous miRNAs that regulate viral life cycle. However, few viral targets have been identified due to the lack of information on KSHV 3' untranslated regions (3'UTRs). Recent genome-wide mapping of KSHV transcripts and 3'UTRs has revealed abundant bicistronic and polycistronic transcripts. The extended 3'UTRs of the 5' proximal genes of bicistronic and polycistronic transcripts offer additional regulatory targets. Indeed, a genome-wide screening of KSHV 3'UTRs has identified several bicistronic and polycistronic transcripts as the novel targets of viral miRNAs. Together, these works have expanded our knowledge of the unique features of KSHV gene regulation program and provided valuable resources for the research community.
    Current opinion in virology. 05/2014; 7C:66-72.

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