Tien-Hsien Chang

Dbp5p, a cytosolic RNA helicase, is required for poly(A) + RNA export

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP‐dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3.

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3ps function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.

Exploring functional relationships between components of the gene expression machinery.

Eukaryotic gene expression requires the coordinated activity of many macromolecular machines including transcription factors and RNA polymerase, the spliceosome, mRNA export factors, the nuclear pore, the ribosome and decay machineries. Yeast carrying mutations in genes encoding components of these machineries were examined using microarrays to measure changes in both pre-mRNA and mRNA levels. We used these measurements as a quantitative phenotype to ask how steps in the gene expression pathway are functionally connected. A multiclass support vector machine was trained to recognize the gene expression phenotypes caused by these mutations. In several cases, unexpected phenotype assignments by the computer revealed functional roles for specific factors at multiple steps in the gene expression pathway. The ability to resolve gene expression pathway phenotypes provides insight into how the major machineries of gene expression communicate with each other.

The current understanding of Ded1p/DDX3 homologs from yeast to human.

DExD/H-box RNA helicases are involved in almost all steps of the eukaryotic mRNA biogenesis. The DEAD-box protein Ded1p/DDX3 is conserved from yeast to human. Various lines of genetic and biochemical evidence have indicated a role of the yeast Ded1p in translation and, most likely, in precursor mRNA splicing as well. In contrast, although recent studies have begun to reveal the function of the mammalian DDX3 in translation control, its exact role remains vague and even controversial. Here, we review these findings and particularly discuss the functional aspects of Ded1p/DDX3 in translation control.

Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

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.

MYBS: a comprehensive web server for mining transcription factor binding sites in yeast

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/.

Roles of Trans and Cis Variation in Yeast Intraspecies Evolution of Gene Expression

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.

A Targeted Bypass Screen Identifies Ynl187p, Prp42p, Snu71p, and Cbp80p for Stable U1 snRNP/Pre-mRNA Interaction

ABSTRACT 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.

Analysis of stress granule assembly in Schizosaccharomyces pombe

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.

Attenuation of 40S Ribosomal Subunit Abundance Differentially Affects Host and HCV Translation and Suppresses HCV Replication

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.