KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphomas. KSHV lytic infection produces PAN RNA, a highly abundant noncoding polyadenylated transcript that is retained in the nucleus. We recently demonstrated that PAN RNA interacts with several viral and cellular factors and can disregulate the expression of genes that modulate immune response. In an effort to define the role of PAN RNA in the context of the virus genome we generated a recombinant BACmid that deleted the PAN RNA locus. Because of the apparent duplication of the PAN RNA locus in BAC36, we generated BAC36CR, a recombinant BACmid that removes the duplicated region. BAC36CR was used as a template to delete most of the PAN RNA locus to generate BAC36CRΔPAN. BAC36CRΔPAN failed to produce supernatant virus and displayed a general decrease in mRNA accumulation of representative immediate early, early and late genes. Most strikingly, K-Rta expression was decreased in lytically induced BAC36CRΔPAN-containing cell lines at early and late time points post induction. Expression of PAN RNA in trans in BAC36CRΔPAN containing cells resulted in an increase in K-Rta expression, however K-Rta over expression failed to rescue BAC36CRΔPAN, suggesting that PAN RNA plays a wider role in virus replication. To investigate the role of PAN RNA in the activation of K-Rta expression, we demonstrate that PAN RNA physically interacts with the ORF50 promoter. RNA chromatin immunoprecipitation assays show that PAN RNA interacts with demethylases JMJD3 and UTX, and the histone methyltransferase MLL2. Consistent with the interaction with demethylases, expression of PAN RNA results in a decrease of the repressive H3K27me3 mark at the ORF50 promoter. These data support a model where PAN RNA is a multifunctional regulatory transcript that controls KSHV gene expression by mediating the modification of chromatin by targeting the KSHV repressed genome

ncFANs: a web server for functional annotation of long non-coding RNAs

Recent interest in the non-coding transcriptome has resulted in the identification of large numbers of long non-coding RNAs (lncRNAs) in mammalian genomes, most of which have not been functionally characterized. Computational exploration of the potential functions of these lncRNAs will therefore facilitate further work in this field of research. We have developed a practical and user-friendly web interface called ncFANs (non-coding RNA Function ANnotation server), which is the first web service for functional annotation of human and mouse lncRNAs. On the basis of the re-annotated Affymetrix microarray data, ncFANs provides two alternative strategies for lncRNA functional annotation: one utilizing three aspects of a coding-non-coding gene co-expression (CNC) network, the other identifying condition-related differentially expressed lncRNAs. ncFANs introduces a highly efficient way of re-using the abundant pre-existing microarray data. The present version of ncFANs includes re-annotated CDF files for 10 human and mouse Affymetrix microarrays, and the server will be continuously updated with more re-annotated microarray platforms and lncRNA data. ncFANs is freely accessible at http://www.ebiomed.org/ncFANs/ or http://www.noncode.org/ncFANs/

Mutation of Rubie, a Novel Long Non-Coding RNA Located Upstream of Bmp4, Causes Vestibular Malformation in Mice

Background: The vestibular apparatus of the vertebrate inner ear uses three fluid-filled semicircular canals to sense angular acceleration of the head. Malformation of these canals disrupts the sense of balance and frequently causes circling behavior in mice. The Epistatic circler (Ecl) is a complex mutant derived from wildtype SWR/J and C57L/J mice. Ecl circling has been shown to result from the epistatic interaction of an SWR-derived locus on chromosome 14 and a C57L-derived locus on chromosome 4, but the causative genes have not been previously identified. Methodology/Principal Findings: We developed a mouse chromosome substitution strain (CSS-14) that carries an SWR/J chromosome 14 on a C57BL/10J genetic background and, like Ecl, exhibits circling behavior due to lateral semicircular canal malformation. We utilized CSS-14 to identify the chromosome 14 Ecl gene by positional cloning. Our candidate interval is located upstream of bone morphogenetic protein 4 (Bmp4) and contains an inner ear-specific, long non-coding RNA that we have designated Rubie (RNA upstream of Bmp4 expressed in inner ear). Rubie is spliced and polyadenylated, and is expressed in developing semicircular canals. However, we discovered that the SWR/J allele of Rubie is disrupted by an intronic endogenous retrovirus that causes aberrant splicing and premature polyadenylation of the transcript. Rubie lies in the conserved gene desert upstream of Bmp4, within a region previously shown to be important for inner ear expression of Bmp4. We found that the expression patterns of Bmp4 and Rubie are nearly identical in developing inner ears

MIR-99a and MIR-99b Modulate TGF-β Induced Epithelial to Mesenchymal Plasticity in Normal Murine Mammary Gland Cells

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs

Autoregulation of the Drosophila Noncoding roX1 RNA Gene

Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. We wanted to find out how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1+ roX2+) females if we provide a heterologous source of MSL2. However, in the absence of roX2, we found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, we found that expression was restored if we introduced either roX2 or a truncated but functional version of roX1. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. We also observed massive cis spreading of the MSL complex from the site of roX1 transcription at its endogenous location on the X chromosome. We propose that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, we found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. We offer a model describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell

Comparative Analysis of Human Protein-Coding and Noncoding RNAs between Brain and 10 Mixed Cell Lines by RNA-Seq

In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome

Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria

Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection

microRNA-122 stimulates translation of hepatitis C virus RNA

Hepatitis C virus (HCV) is a positive strand RNA virus that propagates primarily in the liver. We show here that the liver-specific microRNA-122 (miR-122), a member of a class of small cellular RNAs that mediate post-transcriptional gene regulation usually by repressing the translation of mRNAs through interaction with their 3′-untranslated regions (UTRs), stimulates the translation of HCV. Sequestration of miR-122 in liver cell lines strongly reduces HCV translation, whereas addition of miR-122 stimulates HCV translation in liver cell lines as well as in the non-liver HeLa cells and in rabbit reticulocyte lysate. The stimulation is conferred by direct interaction of miR-122 with two target sites in the 5′-UTR of the HCV genome. With a replication-defective NS5B polymerase mutant genome, we show that the translation stimulation is independent of viral RNA synthesis. miR-122 stimulates HCV translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage. In conclusion, the liver-specific miR-122 may contribute to HCV liver tropism at the level of translation

A Protein Inventory of Human Ribosome Biogenesis Reveals an Essential Function of Exportin 5 in 60S Subunit Export

A systematic search for human ribosome biogenesis factors shows conservation of many aspects of eukaryotic ribosome synthesis with the well-studied process in yeast and identifies an export route of 60S subunits that is specific for higher eukaryotes