Mechanisms of RNA localization and translational regulation.

Transcript localization and translational regulation are two post-transcriptional mechanisms for the spatial and temporal regulation of protein production. During the past year, two transcript localization mechanisms have been elaborated in some detail. Where localization involves directional transport on cytoskeletal tracks, links between the transcripts and the cytoskeletal molecular motors have been elaborated. In the case of localization by generalized transcript degradation combined with localized protection, trans-acting pathways and cis-acting elements for degradation and protection have been identified. A third transcript localization mechanism, vectorial transport out of the nucleus into a particular cytoplasmic domain, was initially thought to localize pair-rule transcripts in Drosophila. However, these have now been shown to be localized by directional transport in the cytoplasm. Transcript localization and translational regulation can be intimately linked in that, for certain messenger RNAs, only the localized fraction of transcripts is translated whereas unlocalized transcripts are translationally repressed. Cis-acting sequences and trans-acting factors that function in translational repression have been identified along with factors involved in relief of translational repression at the site of localization.

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro.

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.

smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo.

nanos mRNA, which encodes the localized component of the Drosophila posterior body patterning determinant, is normally translated only at the posterior pole of the embryo, where the mRNA is concentrated. Here we identify two similar cis-acting sequences in the nanos mRNA 3' untranslated region that mediate translational repression. These sequences bind an embryonic protein of 135 kD, smaug, and we refer to them as smaug recognition elements (SREs). Analysis of point mutations in the SREs reveals a strong correlation between smaug binding and translational repression; mutants unable to bind smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind smaug remain repressed translationally. These results strongly suggest that smaug acts in translational repression of unlocalized nanos mRNA. Translational repression is essential, as embryos expressing a nanos mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of the RNA.

Translational regulation of maternal mRNAs.

Translational regulation of maternal mRNAs serves to constrain their activities in both time and space. Both types of constraint might be expected to be critical for normal development and the rather short list of maternal mRNAs for which this has been shown to be true has expanded considerably over the past year. Substantial progress has also been reported in the identification and characterization of the cis-acting elements and trans-acting factors that mediate translational regulation and their interactions with one another.

Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.

Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.

Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins.

Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.

Herpes simplex virus VP16 forms a complex with the virion host shutoff protein vhs.

Herpes simplex virus (HSV) virions contain at least two regulatory proteins that modulate gene expression in infected cells: the transcriptional activator VP16 and the virion host shutoff protein vhs. VP16 stimulates transcription of the HSV immediate-early genes, and vhs suppresses host protein synthesis and induces accelerated turnover of cellular and viral mRNAs. We report here that vhs binds directly to VP16: vhs and VP16 were coprecipitated from infected cells by an anti-vhs antiserum, and vhs and VP16 protein A fusions each bound intact versions of the other protein in a solid-phase capture assay. In addition, vhs and VP16 interacted in the two-hybrid activator system when coexpressed in Saccharomyces cerevisiae. vhs residues 238 to 344 were sufficient for the interaction, and the VP16 acidic transcriptional activation domain was not required. vhs blocked the ability of VP16 to enter a multiprotein complex on an immediate-early TAATGARATTC consensus sequence, indicating that vhs interacts with one or more regions of VP16 required for promoter recognition. We suggest that this interaction may play a structural role in the assembly of HSV virions and modulate the activity of vhs during infection.

Identification and characterization of the virion-induced host shutoff product of herpes simplex virus gene UL41.

The virion-induced host shutoff product of the herpes simplex virus UL41 gene is required for shutoff of host translation and degradation of cellular mRNAs. We employed a rabbit antipeptide antiserum to identify a 58K UL41-related phosphoprotein in infected cells. We also provide evidence that this protein is a component of the virus particle, consistent with its role in virion-induced shutoff.

Differential regulation of endogenous and transduced beta-globin genes during infection of erythroid cells with a herpes simplex virus type 1 recombinant.

We infected murine erythroleukemia cells with a nondefective herpes simplex virus (HSV) type 1 recombinant bearing the rabbit beta-globin gene under the control of its own promoter, in order to compare the regulation of a cellular gene residing in the viral genome to that of its active endogenous counterpart. We found that the viral globin gene was activated by HSV immediate-early polypeptides, whereas expression of the endogenous beta-globin gene was strongly suppressed: transcription was greatly inhibited, and beta-globin mRNA was rapidly degraded. Degradation of globin mRNA was induced by a component of the infecting virion and required a functional UL41 gene product. These results demonstrate that HSV products can have opposing effects on the expression of homologous genes located in the cellular and viral genomes and suggest that the preferential expression of HSV genes that occurs during infection is not achieved solely through sequence-specific differentiation between viral and cellular promoters or mRNAs.