Abstract
A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma–associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.
Highlights
The nuclear export of messenger RNA (mRNA) composes one part of a larger network of molecular events that begin with transcription of the mRNA in the nucleus and end with its translation and degradation in the cytoplasm
In addition to excising introns, splicing is essential for the recruitment of a several protein complexes to mRNA, one example being the human transcription/export complex, which is required for mRNA export
An intriguing caveat in herpesvirology is that herpesviruses, such as Kaposi’s sarcoma–associated herpesvirus, produce some mRNAs that lack introns and do not undergo splicing
Summary
The nuclear export of mRNA composes one part of a larger network of molecular events that begin with transcription of the mRNA in the nucleus and end with its translation and degradation in the cytoplasm. During trafficking to the cytoplasm, a nascent mRNA undergoes numerous co-transcriptional processing steps, including 59 capping, splicing to remove introns and 39 polyadenylation [1,2,3]. Of these events it has become clear that splicing is important for mRNA nuclear export [4]. Two distinct multi-protein complexes are recruited to cellular mRNAs as a consequence of splicing, namely the human transcription/export complex (hTREX) and the exon-junction complex (EJC). A second multi-protein complex, termed the exon-junction complex (EJC) is deposited
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