For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.

Author Summary

Hepatitis C virus (HCV) infection causes chronic liver diseases that threaten ∼2% of the world population. There is no effective vaccine, and the current standard therapy, the combination of interferon and ribavirin, is effective to less than 50% of genotype-1 infected patients. While antivirals targeting at specific HCV proteins might ultimately lose their effectiveness due to the emergence of resistance-associated mutations, an alternative strategy that explores the genetic stability of host factors indispensable for HCV replication may provide better therapeutic targets for anti-HCV medicine. Here, we employed a loss-of-function screen method to identify such potential targets and uncovered a potential novel anti-HCV mechanism by manipulating the biogenesis of 40S ribosomal subunit. We showed that inhibiting 40S ribosome biogenesis can selectively suppress HCV translation and thus effectively inhibit HCV replication. In contrast to the conventional thinking, the 40S ribosomal subunit can differentially affect different translational modes, and HCV translation is more sensitive to the amounts of 40S ribosomal subunit as compared to general translation in host cell. Since HCV is known to evade anti-viral effects including translational suppression elicited by interferon, our findings may help design a therapeutic strategy to supplement interferon-based therapy and minimize mutation-associated drug resistance problem.

Both cis and trans mutations contribute to gene expression divergence within and between species. We used Saccharomyces cerevisiae as a model organism to estimate the relative contributions of cis and trans variations to the expression divergence between a laboratory (BY) and a wild (RM) strain of yeast. We examined whether genes regulated by a single transcription factor (TF; single input module, SIM genes) or genes regulated by multiple TFs (multiple input module, MIM genes) are more susceptible to trans variation. Because a SIM gene is regulated by a single immediate upstream TF, the chance for a change to occur in its trans-acting factors would, on average, be smaller than that for a MIM gene. We chose 232 genes that exhibited expression divergence between BY and RM to test this hypothesis. We examined the expression patterns of these genes in a BY–RM coculture system and in a BY–RM diploid hybrid. We found that trans variation is far more important than cis variation for expression divergence between the two strains. However, because in 75% of the genes studied, cis variation has significantly contributed to expression divergence, cis change also plays a significant role in intraspecific expression evolution. Interestingly, we found that the proportion of genes with diverged expression between BY and RM is larger for MIM genes than for SIM genes; in fact, the proportion tends to increase with the number of transcription factors that regulate the gene. Moreover, MIM genes are, on average, subject to stronger trans effects than SIM genes, though the difference between the two types of genes is not conspicuous.

To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5′ splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5′ splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5′ splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.

Correct interactions between transcription factors (TFs) and their binding sites (TFBSs) are of central importance to gene regulation. Recently developed chromatin-immunoprecipitation DNA chip (ChIP-chip) techniques and the phylogenetic footprinting method provide ways to identify TFBSs with high precision. In this study, we constructed a user-friendly interactive platform for dynamic binding site mapping using ChIP-chip data and phylogenetic footprinting as two filters. MYBS (Mining Yeast Binding Sites) is a comprehensive web server that integrates an array of both experimentally verified and predicted position weight matrixes (PWMs) from eleven databases, including 481 binding motif consensus sequences and 71 PWMs that correspond to 183 TFs. MYBS users can search within this platform for motif occurrences (possible binding sites) in the promoters of genes of interest via simple motif or gene queries in conjunction with the above two filters. In addition, MYBS enables users to visualize in parallel the potential regulators for a given set of genes, a feature useful for finding potential regulatory associations between TFs. MYBS also allows users to identify target gene sets of each TF pair, which could be used as a starting point for further explorations of TF combinatorial regulation. MYBS is available at http://cg1.iis.sinica.edu.tw/~mybs/.

Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus–host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.

URH49 is a mammalian protein that is 90% identical to the DExH/D box protein UAP56, an RNA helicase that is important for splicing and nuclear export of mRNA. Although Saccharomyces cerevisiae and Drosophila express only a single protein corresponding to UAP56, mRNAs encoding URH49 and UAP56 are both expressed in human and mouse cells. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), indicating that both proteins have similar functions. UAP56 mRNA is more abundant than URH49 mRNA in many tissues, although in testes URH49 mRNA is much more abundant. UAP56 and URH49 mRNAs are present at similar levels in proliferating cultured cells. However, when the cells enter quiescence, the URH49 mRNA level decreases 3–6-fold while the UAP56 mRNA level remains relatively constant. The amount of URH49 mRNA increases to the level found in proliferating cells within 5 h when quiescent cells are growth-stimulated or when protein synthesis is inhibited. URH49 mRNA is relatively unstable (T½ = 4 h) in quiescent cells, but is stabilized immediately following growth stimulation or inhibition of protein synthesis. In contrast, there is much less change in the content or stability of UAP56 mRNA following growth stimulation. Our observations suggest that in mammalian cells, two UAP56-like RNA helicases are involved in splicing and nuclear export of mRNA. Differential expression of these helicases may lead to quantitative or qualitative changes in mRNA expression.

The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution.

The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination – which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1–5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.

Background

The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA.

Results

Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality.

Conclusion

These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.