Cooperative Interaction of Transcription Termination Factors with the RNA Polymerase II C-terminal Domain

Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Serine 2 phosphorylation density is highest

Unphosphorylated SR-Like Protein Npl3 Stimulates RNA Polymerase II Elongation

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation

The Set3/Hos2 Histone Deacetylase Complex Attenuates cAMP/PKA Signaling to Regulate Morphogenesis and Virulence of Candida albicans

Candida albicans, like other pleiomorphic fungal pathogens, is able to undergo a reversible transition between single yeast-like cells and multicellular filaments. This morphogenetic process has long been considered as a key fungal virulence factor. Here, we identify the evolutionarily conserved Set3/Hos2 histone deacetylase complex (Set3C) as a crucial repressor of the yeast-to-filament transition. Cells lacking core components of the Set3C are able to maintain all developmental phases, but are hypersusceptible to filamentation-inducing signals, because of a hyperactive cAMP/Protein Kinase A signaling pathway. Strikingly, Set3C-mediated control of filamentation is required for virulence in vivo, since set3Δ/Δ cells display strongly attenuated virulence in a mouse model of systemic infection. Importantly, the inhibition of histone deacetylase activity by trichostatin A exclusively phenocopies the absence of a functional Set3C, but not of any other histone deacetylase gene. Hence, our work supports a paradigm for manipulating morphogenesis in C. albicans through alternative antifungal therapeutic strategies

Serological Profiling of a Candida albicans Protein Microarray Reveals Permanent Host-Pathogen Interplay and Stage-Specific Responses during Candidemia

Candida albicans in the immunocompetent host is a benign member of the human microbiota. Though, when host physiology is disrupted, this commensal-host interaction can degenerate and lead to an opportunistic infection. Relatively little is known regarding the dynamics of C. albicans colonization and pathogenesis. We developed a C. albicans cell surface protein microarray to profile the immunoglobulin G response during commensal colonization and candidemia. The antibody response from the sera of patients with candidemia and our negative control groups indicate that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans. This report also identifies cell surface antigens that are specific to different phases (i.e. acute, early and mid convalescence) of candidemia. We identified a set of thirteen cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Interestingly, a large proportion of these cell surface antigens are involved in either oxidative stress or drug resistance. In addition, we identified 33 antigenic proteins that are enriched in convalescent sera of the candidemia patients. Intriguingly, we found within this subset an increase in antigens associated with heme-associated iron acquisition. These findings have important implications for the mechanisms of C. albicans colonization as well as the development of systemic infection

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila

We thank our colleagues Linda Thompson and Luke Szweda at OMRF, and Gary Struhl at Columbia University for critical comments on the manuscript. We are grateful to Rolf Bodmer (Sanford-Burnham-Presby Medical Discovery Institute), Joquim Culi (CSIC-UPO), Susan Abmayr (Stowers Institute) and Laurent Perrin (TAGC) for fly stocks and antibodies. We also thank the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the TRiP at Harvard Medical School for fly stocks. We acknowledged the Imaging Core Facility at OMRF for excellent technical assistance.Author Summary The heart is increasingly recognized to serve an important role in the regulation of whole-body lipid homeostasis; however, the underlying mechanisms remained poorly understood. Here, our study in Drosophila reveals that cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body (insect liver and adipose tissue). We found that apoB-lipoproteins generated by the Drosophila cardiomyocytes serve an equally significant role as their fat body-derived counterparts in maintaining systemic lipid homeostasis on normal food diet. Importantly, on high fat diet (HFD), the cardiomyocyte-derived apoB-lipoproteins are the major determinants of whole-body lipid metabolism, a role which could be attributed to the HFD-induced up-regulation of apoB-lipoprotein biosynthesis genes in the cardiomyocytes and their down-regulation in the fat body. Taken together, our results reveal that apoB-lipoproteins are new players in mediating the heart control of lipid metabolism, and provide first evidence supporting the notion that HFD-induced differential regulation of apoB-lipoprotein biosynthesis genes could alter the input of different tissue-derived apoB-lipoproteins in systemic lipid metabolic control.Yeshttp://www.plosgenetics.org/static/editorial#pee

Transcriptome 3′end organization by PCF11 links alternative polyadenylation to formation and neuronal differentiation of neuroblastoma

In gene regulation, diversification at the transcriptome 3′end is linked to differentiation and dedifferentiation. Here, the authors discover extensive transcriptome 3′end-alterations in neuroblastoma, regulated by PCF11, and provide an interactive data repository of transcriptome-wide alternative polyadenylation

Nascent transcription from the nmt1 and nmt2 genes of Schizosaccharomyces pombe overlaps neighbouring genes.

We have determined the extent of the primary transcription unit for the two highly expressed genes nmt1 and nmt2 of Schizosaccharomyces pombe. Transcription run-on analysis in permeabilized yeast cells was employed to map polymerase density across the 3'-flanking region of these two genes. Surprisingly, polymerases were detected 4.3 kb beyond the nmt1 polyadenylation [poly(A)] site and 2.4 kb beyond the nmt2 poly(A) site, which in each case have transcribed through an entire convergent downstream transcription unit. However, the steady-state levels of both downstream genes were unaffected by the high level of nmt1 or nmt2 nascent transcription. Analysis of nmt1 and nmt2 RNA 3' end formation signals indicates that efficient termination of transcription requires not only a poly(A) signal but also additional pause elements. The absence of such pause elements close to the poly(A) sites of these genes may account for their extended nascent transcripts