The diversity of plasma membrane (PM) proteins presents a challenge for the achievement of cargo-specific regulation of endocytosis. Here, we describe a family of proteins in yeast (ARTs, for arrestin-related trafficking adaptors) that function by targeting specific PM proteins to the endocytic system. Two members (Art1 and Art2) of the family were discovered in chemical-genetic screens, and they direct downregulation of distinct amino acid transporters triggered by specific stimuli. Sequence analysis revealed a total of nine ART family members in yeast. In addition to similarity to arrestins, the ARTs each contain multiple PY motifs. These motifs are required for recruitment of the Rsp5/Nedd4-like ubiquitin ligase, which modifies the cargoes as well as the ARTs. As a result, ubiquitinated cargoes are internalized and targeted to the vacuole (lysosome) for degradation. We propose that ARTs provide a cargo-specific quality-control pathway that mediates endocytic downregulation by coupling Rsp5/Nedd4 to diverse plasma membrane proteins.
We have identified a virus-activated factor (VAF) that binds to a regulatory element shared by different virus-inducible genes. We provide evidence that VAF contains two members of the interferon regulatory factor (IRF) family of transcriptional activator proteins (IRF-3 and IRF-7), as well as the transcriptional coactivator proteins p300 and CBP. Remarkably, VAF, as well as recombinant IRF-3 and IRF-7 proteins, binds very weakly to the interferon-beta (IFN-beta) gene promoter in vitro. However, in virus-infected cells, both proteins are recruited to the endogenous IFN-beta promoter as part of a protein complex that includes ATF-2/c-Jun and NF-kappa B. These observations provide a unique example of the coordinate activation of multiple transcriptional activator proteins and their highly cooperative assembly into a transcriptional enhancer complex in vivo.
Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.development | expression T he non-protein-coding portion of the mammalian genome is transcribed into a vast array of RNA species (1), some of which play important roles in cellular regulation, development, and disease (2). The long noncoding RNAs (lncRNAs) are of particular interest because they are known to contribute to gene silencing (3), X inactivation (4), imprinting (5, 6), and development (7-9), but there is limited understanding of the genomic origin, regulation, and function of lncRNA molecules in individual cell types.Embryonic stem cells (ESCs) are widely used as a model system to study transcriptional control of cell state during early development (10-13), yet there is no catalog of lncRNAs in human (h) ESCs, and it is not clear how lncRNAs are regulated in these cells. Catalogs of lncRNAs have been recently described in various murine (14, 15) and human cell types (16)(17)(18)(19), but the majority were limited to spliced lncRNA species (14-16, 18) and those distant from protein-coding genes (14-17). Because lncRNAs tend to be cell-type-specific (16, 18), these catalogs likely contain only a very small fraction of lncRNAs expressed in hESCs.We describe here catalogs of human and murine ESC lncRNAs and the genomic regions from which these RNA species arise. We find that the majority of these lncRNAs originate from divergent transcription of lncRNA/mRNA gene pairs and that many such gene pairs are coordinately regulated when ESCs differentiate.Results lncRNAs Expressed in Human ESCs. We compiled a catalog of lncRNA species expressed in hESCs as summarized in Fig. 1A. An initial pool of RNA candidates was generated by sequencing polyadenylated RNA species from hESCs and supplementing these with EST data from the full-length long Japan (FLJ) collection of sequenced human cDNAs, which contains transcripts expressed in >60 human tissues, including embryonal tissue (20). An initial pool of 170,162 ncRNA candidates (Dataset S1) was obtained after removing protein-coding transcripts based on the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq). This pool was further filtered by using multiple criteria to identify lncRNAs. The RNA species were required to have a 5′ end that originates from a genomic site where there is corroborating evidence of active transcript...
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