ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervious system

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. Here, we present evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. Our study provides evidence for a regulatory link between promoter-proximal pausing and APA

Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3.

We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses

Generation of Transgenic Mice to Evaluate Promoter Activity and Specificity of Two Human Endogenous Retrovirus Long Terminal Repeats = Untersuchungen zur Promotor-Aktivität und -Spezifität von zwei Long Terminal Repeats humaner endogener Retroviren in transgenen Mäusen

Generation of Transgenic Mice to Evaluate Promoter Activity and Specificity of two Human Endogenous Retrovirus Long Terminal Repeats Human Endogenous Retrovirus Long Terminal Repeats (HERV-LTRs) comprise 1.8% of the human genome (52.7 Mb). These sequences contain all the signal structures necessary for the regulation of gene transcription, such as promoters, enhancers and transcription factor binding sites. There is evidence that HERV-LTRs regulate gene expression in tissue-specific manner. This potential could be used to drive the expression of therapeutic genes, delivered by retroviral vector systems, in a safe and efficient manner. The HERV-H-H6 LTR and the HERV-L LTR were chosen for the generation of transgenic mice. Their promoter activity and specificity had prior been tested in a luciferase expression vector in vitro (Schoen et al., 2001). HERV-L was cloned into a luciferase expression vector and HERV-H-H6 was inserted into a enhanced green fluorescent protein (EGFP) expression vector. Transgenic mice were generated by DNAmicroinjection into pronuclei of zygotes. One pBL-HERV-L transgenic line and four pEGFP-HERV-H-H6 transgenic lines were established and analyzed. While the HERV-L promoter was not active in transgenic animals, pEGFP-HERV-H-H6 was expressed in gonads of mice of two transgenic lines. As only a single, non-expressing transgenic line was available, HERV-L promoter activity and specificity could not be evaluated. Additional transgenic lines have to be established. Expression level and pattern of the HERV-H-H6 promoter indicate specificity for gonad tissue. Whether the HERV-H-H6 promoter activity is linked to steroid production in cells remains to be clarified. Evaluating promoter activity in transgenic mice in two different expression vectors is not exclusively about the promoters, but also involves knowledge about the reporter genes. Advantages and limits of current applications of both luciferase and EGFP (with focus on the EGFP gene) are described in REVIEW OF THE LITERATURE. The conjunction of EGFP with the HERV-H-H6 promoter is to be seen critically, as all published methods for detection of EGFP in mice are described with EGFP linked to strong promoters. Problems like autofluorescence in fluorescence microscopy might be encountered when weaker promoters, such as HERV-LTRs, drive EGFP expression.Untersuchungen zur Promotor-Aktivität und –Spezifität von zwei Long Terminal Repeats humaner endogener Retroviren in transgenen Mäusen 1.8% des humanen Genoms bestehen aus Long Terminal Repeats Humaner Endogener Retroviren (HERV-LTRs). Solche Sequenzen enthalten alle Strukturen, die für die Regulierung von Transkription benötigt werden: Promotoren, Enhancer and Bindungsstellen für Transkriptionsfaktoren. Es gibt Hinweise, daß HERV-LTRs die Expression von Genen gewebespezifisch regulieren können. Eingebaut in retrovirale Genfähren, könnten HERV-LTRs therapeutische Gene sicher und effizient aktivieren. Zur Generierung transgener Mäuse wurden der HERV-H-H6 LTR und der HERV-L LTR ausgewählt. Deren Promoter Eigenschaften, wie Aktivität und Gewebespezifität, waren bereits in vitro untersucht worden (Schoen et al., 2001). Der HERV-L LTR wurde in einen Luciferase Expressionsvektor und der HERV-H-H6 LTR in einen Enhanced Green Fluorescent Protein (EGFP) Expressionsvektor kloniert. Transgene Mäuse enstanden durch DNA-Mikroinjektion in den Vorkern von Zygoten. Eine pBL-HERV-L transgene Linie und vier pEGFP-HERV-H-H6 transgene Linien wurden gezüchtet und auf Integration sowie Expression der Genkonstrukte untersucht. Während der HERV-L Promoter keine Aktivität zeigte, war Expression von pEGFP-HERV-H-H6 in Keimdrüsen von Mäusen aus zwei transgenen Linien nachweisbar. Da für das Genkonstrukt pBL-HERV-L nur eine einzige, nicht-exprimierende transgene Linie aufgebaut werden konnte, können keine Aussagen über die Aktivität und Gewebespezifität des HERV-L Promoters getroffen werden. Zu diesem Zwecke müssten weitere pBL-HERV-L transgene Linien untersucht werden. Das Expressionsmuster des pEGFP-HERV-H-H6 Genkonstruktes, weißt auf eine mögliche Gewebespezifität für Keimdrüsen hin. Eine eventuelle Verknüpfung der Aktivität des HERV-H-H6 LTRs mit der Produktion von Steroidhormonen müsste weitergehend geklärt werden.Da in dieser Arbeit zwei unterschiedliche Reportergen Systeme in der Maus angewandt wurden, sind im Literaturteil Vorteile und Einschränkungen von aktuellen Nachweisverfahren beider Reportergene, mit Schwerpunkt EGFP, in Mausgewebe zusammengefasst. Die Verbindung von EGFP mit dem HERV-H-H6 Promoter ist als kritisch zu beurteilen: Alle beschriebenen Nachweisverfahren für EGFP in der Maus gründen auf Mausmodellen, in denen das EGFP von einem starken Promoter kontrolliert wurde. Bei potenziell schwächeren Promotoren, wie HERV-LTRs, können Probleme auftreten, wie z.B. Autofluoreszenz bei der Fluoreszenzmikroskopie

Ab Initio Identification of Novel Regulatory Elements in the Genome of Trypanosoma brucei by Bayesian Inference on Sequence Segmentation

Background: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes. Methodology/Principle Findings: Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site. Conclusions/Significance: The nature of the elements discovered reinforces the hypothesis that context dependant RN

Control of human papillomavirus gene expression by alternative splicing

Human papillomaviruses possess circular double stranded DNA genomes of around 8 kb in size from which multiple mRNAs are synthesized during an infectious life cycle. Although at least three viral promoters are used to initiate transcription, viral mRNAs are largely the product of processing of pre-mRNAs by alternative splicing and polyadenylation. The HPV life cycle and viral gene expression are tightly linked to differentiation of the epithelium the virus infects: there is an orchestrated production of viral mRNAs and proteins. In this review we describe viral mRNA expression and the roles of the SR and hnRNP proteins that respectively positively and negatively regulate splicing. We discuss HPV regulation of splicing factors and detail the evidence that the papillomavirus E2 protein has splicing-related activities. We highlight the possibility that HPV-mediated control of splicing in differentiating epithelial cells may be necessary to accomplish the viral replication cycle

ISOLATION AND CHARACTERIZATION OF THE FOUR ARABIDOPSIS THALIANA POLY(A) POLYMERASE GENES

Poly(A) tail addition to pre-mRNAs is a highly coordinated and essential step in mRNA maturation involving multiple cis- and trans-acting factors. The trans-acting factor, poly(A) polymerase (PAP) plays an essential role in the polyadenylation of mRNA precursors. The Arabidopsis thaliana genome contains four putative PAP genes. We have found, using in silico analysis and transgenic plants expressing GUS under the control of the four PAP promoters, that each of these genes is expressed in overlapping, yet unique patterns. This gives rise to the possibility that these genes are not redundant and may be essential for plant survival. To further test this, inducible RNAi and T-DNA mutagenized plants were obtained and analyzed. Plants lacking all, or most, of each PAP gene product, due to RNAi induction, were not viable at any of the stages of plant growth tested. Furthermore, T-DNA PCR analysis determined that no plants containing a homozygous mutation, were viable. This data reveals that lack of any of the four PAP gene products has a significant effect on the plants ability of survive, thus indicating that each PAP gene is essential. Finally, transient expression experiments with each of the full length PAP cDNAs fused to GFP showed that the PAP I, PAP II and PAP IV gene products are localized throughout the nucleus and within nuclear speckles. The cellular localization of PAP III could not be determined

Functional characterization of BRD4 in transcription elongation and termination

The eukaryotic transcription machinery consisting of RNA polymerase II (Pol II) and a small number of minimally required initiation, elongation and termination factors is well-characterized structurally and biochemically, while functional characterization traditionally has been hampered by the limitations of the available perturbation strategies and readouts. In addition, knowledge of accessory transcription regulators is likely incomplete. Proteins of the conserved bromodomain and extra-terminal domain (BET) family have been demonstrated to function in maintaining normal transcription genome-wide by promoting elongation. However, the underlying molecular mechanism and also the specific contribution of each of the protein family members remained elusive. Moreover, it was unclear if BET proteins serve additional direct or indirect functions in transcription or transcription-coupled processes. To address these aspects, this study combines targeted protein degradation, a new strategy to near-completely deplete a protein of interest within minutes to hours, with complementary transcriptome-, genome- and proteome-wide readouts of high temporal or spatial resolution. It provides evidence that specifically BRD4 is involved in transcription control, although BRD2 and BRD3 might have partially overlapping functions. Within 120 minutes, BRD4-selective degradation results in a global reduction of elongating Pol II from the gene body of most protein-coding and long non-coding RNA genes, as can be detected by native elongating transcript sequencing with spike-in normalization (SI-NET-seq). Concomitantly, Pol II accumulates in the promoter-proximal region, suggesting a defect in promoter-proximal pause release. Profiling elongation factors upon BRD4 depletion reveals a decrease in occupancy, suggesting an assembly defect of the active elongation complex. In particular, PAF1 binding is decreased throughout the transcribed region. Unexpectedly, BRD4-selective degradation also induces a severe transcription termination defect at a subset of genes. Particularly, Pol II continues transcribing on average three kilobases beyond its usual termination zone, which is accompanied by inefficient cleavage of the nascent transcript at the pA site. Chromatin immunoprecipitation with sequencing (ChIP-seq) reveals a significant reduction of RNA 3’ end processing factors of the CPSF and CstF modules near the 3’ gene end as well as in the promoter-proximal and the gene body region. The failure to recruit RNA 3’ end processing as well as PAF subunits, SPT5 and SPT6, but not CDK9, could be independently confirmed by quantitative mass spectrometry. Co-immunoprecipitation experiments indicate that the recruitment of 3’ processing factors either directly depends on BRD4 or could be mediated by elongation factors. Also, BRD4-selective degradation locally increases the binding of Pol II and SPT5 at transcribed enhancers, whereas enhancer accessibility appears to be not BRD4-dependent. Altogether, the data establish a role of BRD4 in coordinating the recruitment of elongation and RNA 3’ end processing factors during an early step of transcription.Die eukaryotische Transkriptionsmaschinerie — insbesondere RNA-Polymerase II (Pol II) und eine überschaubare Gruppe essentieller Initiation-, Elongations- und Terminationsfaktoren — ist strukturell als auch biochemisch gut charakterisiert, wohingegen die funktionelle Charakterisierung durch das Fehlen geeigneter Pertubationsmethoden und sensitiver experimenteller Analyseverfahren lange erschwert wurde. Auch sind viele nicht-essentielle Transkriptionsregulatoren wahrscheinlich noch unbekannt. Es konnte gezeigt werden, dass die durch zwei Bromodomänen und eine sog. extra-terminale Domäne gekennzeichneten Proteine der konservierten BET-Proteinfamilie nötig sind, die normale Transkriptionsaktivität der Zelle — vermutlich durch Regulation der Elongation — aufrecht zu erhalten. Unklar sind jedoch der molekulare Mechanismus als auch die Rolle der einzelnen Proteine der Proteinfamilie. Darüber hinaus ist denkbar, dass BET-Proteine direkt oder indirekt auch an weiteren transkriptionellen oder co-transkriptionellen Prozessen beteiligt sind. Die genannten Aspekte adressiert die vorliegende Studie einerseits durch die Verwendung eines relativ neuen Verfahrens zur gezielten und schnellen Degradierung eines Proteins — in diesem Fall BRD4 — und andererseits mit komplementären transkriptom-, genom- und proteomweiten Methoden, die eine hohe zeitliche oder räumliche Auflösung bieten. Auf diese Weise kann gezeigt werden, dass die Transkription in hohem Maße von BRD4 abhängt, obgleich nicht auszuschließen ist, dass BRD2 oder BRD3 eine ähnliche Funktion haben. So belegt SI-NET-seq, eine spike-in-gestützten Methode zur quantitativen Analyse naszenter RNA unter nativen Bedingungen, dass die gezielte Degradierung von BRD4 innerhalb von 120 Minuten zur globalen Verringerung transkribierender Pol II am Genkörper der meisten proteinkodierenden und langen nicht-kodierenden RNA-Gene führt. Gleichzeitig akkumuliert transkribierende Pol II in der promotor-proximalen Region, was auf einen Defekt am Übergang von der promotor-proximalen Pause zur produktiven Elongation hindeutet. Einen Hinweis auf eine Funktion von BRD4 bei der Assemblierung des Elongationskomplexes liefert die Beobachtung, dass die Chromatinbindung von Elongationsfaktoren nach BRD4-Degradierung verringert ist. Insbesondere PAF1 zeigt eine Abnahme in allen Genbereichen. Unerwarteterweise führt die gezielte Degradierung von BRD4 bei einer großen Gruppe von Genen auch zu einem Terminationsdefekt — im Durchschnitt erfolgt die Termination drei Kilobasen später als unter Kontrollbedingungen — und vermindert die Effizient, mit der die naszente RNA an der Polyadenylierungsstelle enzymatisch geschnitten wird. Tatsächlich binden CPSF- und CstF-Faktoren der 3'-RNA-Prozessierungsmaschinerie nach BRD4-Degradierung signifikant weniger am 3'-Ende der Gene, aber auch im promotor-proximalen Bereich und am Genkörper, wie mittels Chromatinimmunpräzipitation (ChIP) gezeigt werden kann. Eine signifikante Abnahme von 3'-Prozessierungsfaktoren sowie von PAF-Untereinheiten, SPT5 und SPT6, nicht aber von CDK9, ist auch durch quantitative Massenspektrometrie nachweisbar. Zudem legen Co-Immunpräzipitationsexperimente nahe, dass 3‘-RNA-Prozessierungsfaktoren entweder direkt durch BRD4 oder indirekt über Elongationsfaktoren rekrutiert werden können. Außerdem korreliert die BRD4-spezifische Degradierung mit der lokalen Zunahme von Pol II und SPT5 an transkribierten Enhancer-Regionen; die Zugänglichkeit des Chromatins gegenüber Tn5-Transposase erscheint hingegen nicht BRD4-abhänging zu sein. Zusammenfassend legen unsere Daten nahe, dass BRD4 zu einem relativ frühen Zeitpunkt des Transkriptionszyklus' die Rekrutierung von Elongations-, aber auch von RNA 3'-Prozessierungsfaktoren koordiniert