In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, we isolated the ASH1 locasome and characterized the associated proteins by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p-bound PUF consensus sequences in the 3 UTR of ASH1 mRNA and repressed the translation of ASH1 mRNA both in vivo and in vitro. In the puf6⌬ strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. We propose that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed.

In Saccharomyces cerevisiae, Ash1p is a speci®c repressor of transcription that localizes exclusively to daughter cell nuclei through the asymmetric localization of ASH1 mRNA. This localization requires four cis-acting localization elements located in the ASH1 mRNA, ®ve trans-acting factors, one of which is a myosin, and the actin cytoskeleton. The RNA-binding proteins that interact with these cis-elements remained to be identi®ed. Starting with the 3¢ most localization element of ASH1 mRNA in the threehybrid assay, element E3, we isolated a clone corresponding to the C-terminus of She3p. We also found that She3p and She2p interact, and this interaction is essential for the binding of She3p with element E3 in vivo. Moreover, She2p was observed to bind the E3 RNA directly in vitro and each of the ASH1 cis-acting localization elements requires She2p for their localization function. By tethering a She3p±MS2 fusion protein to a reporter RNA containing MS2 binding sites, we observed that She2p is dispensable for She3p±MS2-dependent RNA localization.

The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47–amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.

Here we report the isolation and characterization of mouse testicular cDNAs encoding the mammalian homologue of the Xenopus germ cell-specific nucleic acid-binding protein FRGY2 (mRNP3+4), hereafter designated MSY2. MSY2 is a member of the Y box multigene family of proteins; it contains the cold shock domain that is highly conserved among all Y box proteins and four basic/aromatic islands that are closely related to the other known germline Y box proteins from Xenopus, FRGY2, and goldfish, GFYP2. Msy2 undergoes alternative splicing to yield alternate N-terminal regions upstream of the cold shock domain. Although MSY2 is a member of a large family of nucleic acid-binding proteins, Southern blotting detects only a limited number of genomic DNA fragments, suggesting that Msy2 is a single copy gene. By Northern blotting and immunoblotting, MSY2 appears to be a germ cell-specific protein in the testis. Analysis of Msy2 mRNA expression in prepubertal and adult mouse testes, and in isolated populations of germ cells, reveals maximal expression in postmeiotic round spermatids, a cell type with abundant amounts of stored messenger ribonucleoproteins. In the ovary, MSY2 is present exclusively in diplotene-stage and mature oocytes. MSY2 is maternally inherited in the one-cell-stage embryo but is not detected in the late two-cell-stage embryo. This loss of MSY2 is coincident with the bulk degradation of maternal mRNAs in the two-cell embryo.

Translational repression during mRNA transport is essential for spatial restriction of protein production. In the yeast Saccharomyces cerevisae, silencing of ASH1 mRNA before it is localized to the bud cortex in late anaphase is critical for asymmetric segregation of Ash1p to the daughter cell nucleus. Puf6p, an ASH1 mRNA-binding protein, has been implicated in this process as a translational repressor, but the underlying mechanism is unknown. Here, we used yeast extract-based in vitro translation assays, which recapitulate translation and phosphorylation, to characterize the mechanism of Puf6p-mediated translational regulation. We report that Puf6p interferes with the conversion of the 48S complex to the 80S complex during initiation, and this repression by Puf6p is mediated through the general translation factor eIF5B (Fun12p in S. cerevisiae). RNA localization is a fundamental mechanism to restrict protein expression to a specific region in the cell, vital to the establishment of cellular polarity and determination of cell fate (St Johnston 2005;Corral-Debrinski 2007;Du et al. 2007). In budding yeast Saccharomyces cerevisiae, ASH1 mRNA localization is required for mating-type switching. The ASH1 transcripts are localized at the bud cortex in late anaphase, which confines the Ash1 protein to the daughter cell nucleus (Long et al. 1997;Takizawa et al. 1997). ASH1 mRNA localization is achieved by active transport along actin bundles (Long et al. 1997;Takizawa et al. 1997) by a core localization complex (the "locasome") consisting of proteins She1/ Myo4, She2, and She3 (Chartrand et al. 2001;Kwon and Schnapp 2001;Darzacq et al. 2003). She2p is the primary RNA-binding protein that recognizes four cis localization elements (E1, E2A, E2B, and E3) on the ASH1 transcript (Chartrand et al. 1999). She2p recruits Myo4p, a type V myosin, to the ASH1 mRNA via the adaptor protein She3p (Bohl et al. 2000;Long et al. 2000;. ASH1 mRNA localization in budding yeast serves as a model to study RNA transport and localization in mammals and other species (Darzacq et al. 2003;St Johnston 2005). Puf6p interacts withTo achieve spatial and temporal regulation of ASH1 expression, translational repression is coordinated during RNA transport to prevent premature protein synthesis. Both cis-and trans-factors have been shown to play critical roles in translational repression during ASH1 mRNA transport. There are four elements in the coding region of ASH1 mRNA that have been proposed to slow down translation during mRNA transport and prevent premature translation of ASH1 (Chartrand et al. 1999(Chartrand et al. , 2002. Two RNA-binding proteins, Khd1p and Puf6p, have been identified that are required for the localization and translation of ASH1 mRNA (Irie et al. 2002;Gu et al. 2004;Paquin et al. 2007). Release of translational repression is needed once ASH1 mRNA localizes and has been implicated in proper ASH1 mRNA anchoring at the bud

ObjectiveCurrent non-invasive early detection of colorectal cancer (CRC) requires improvement. We aimed to identified a fecal Clostridium symbiosum-based biomarker for early and advanced colorectal cancer detection.DesignIn the test stage, the relative abundance of Clostridium symbiosum (C. symbiosum) was measured by qPCR in 781 cases including 242 controls, 212 colorectal adenoma (CRA) patients, 109 early CRC (tumor restricted to the submucosa) patients, 218 advanced CRC patients. The prediction accuracy was compared to Fusobacterium nucleatum (F. nucleatum), fecal immunochemical test (FIT) and CEA (carcinoembryonic antigen) and validated in an independent cohort of 256 subjects. Current status of the trial:ongoing/still enrolling. Primary endpoint:June, 2017 (Clinicaltrials.gov Identifier NCT02845973).ResultsSignificant stepwise increase of C. symbiosum abundance was found in CRA, early CRC and advanced CRC (P < 0.01). C. symbiosum outperformed all the other markers in early CRC prediction performance. The combination of C. symbiosum and FIT achieved better performance (0.803 for test cohort and 0.707 for validation cohort). For overall discrimination of CRCs, the combination of all above markers achieved the performance of 0.876.ConclusionsFecal C. symbiosum is a promising biomarker for early and noninvasive detection of colorectal cancer, being more effective than F. nucleatum, FIT and CEA. Combining C. symbiosum and FIT or CEA may improve the diagnosis power.

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3′-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3′-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

SummaryMetastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, b-actin, a-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.