Abstract: A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3'end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.
Abstract: The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogenetic analysis of the Gli3 locus that uncovered a novel regulatory element, HCNE1. HCNE1 contains a compound Pbx/Meis binding site that binds Pbx and Meis/Prep proteins in vitro and in vivo. We show that HCNE1 recapitulates Gli3 expression in the developing neural tube and that mutations in the Pbx/Meis binding site affect the spatiotemporal control of HCNE1 transcriptional activity. Ectopic expression or loss of function of Pbx and Meis/Prep proteins in the chick and mouse embryo results in aberrant expression of endogenous Gli3 transcripts. We propose a novel role for TALE proteins in establishing the correct spatiotemporal expression pattern of Gli3 in the vertebrate spinal cord, thus implicating TALE transcription factors in early embryonic patterning events controlled by Sonic hedgehog signaling.
Abstract: The TALE family of homeodomain containing transcription factors consists of the Meis, Prep and Tgif, and the Pbx subfamily of proteins. Several TALE orthologues have been identified in amniotes, but no comprehensive analysis of their expression pattern during embryogenesis has been performed. Here, we report on TALE gene expression in the avian embryo. During embryonic development, Pbx genes are predominantly expressed in the neural ectoderm and paraxial mesoderm, although Pbx3 is restricted to the intermediate and lateral mesoderm, and anterior central nervous system. Members of the Meis, Prep, and Tgif subfamilies are expressed at high levels in the paraxial mesoderm, and display differential expression along the anterior-posterior and dorsoventral axes of the developing neural tube. Overall the expression patterns reported in this study are consistent with the known function of the TALE gene family in controlling early patterning of limb, neural tube and paraxial mesoderm tissues during embryogenesis.
Abstract: Heterochromatic silencing is important for repressing gene expression, protecting cells against viral invasion, maintaining DNA integrity and for proper chromosome segregation. Recently, it has become apparent that expression of eukaryotic genomesis far more complex than had previously been anticipated. Strikingly, it has emerged that most of the genome is transcribed including intergenic regions and heterochromatin, calling for us to re-address the question of how gene silencing is regulated and re-evaluate the concept ofheterochromatic regions of the genome being transcriptionally inactive. Although heterochromatic silencing can be regulated at the transcriptional level, RNA degrading activities supplied either by the exosome complex or RNAi also significantly contribute to this process. The exosome also regulates noncoding RNAs (ncRNAs) involved in the establishment of heterochromatin, further underscoring its role as the major cellular machinery involved in RNA processing and turn-over. This multilevel control of the transcriptome may be utilized to ensure greater accuracy of gene expression and allow distinction between functional transcripts and background noise. In this chapter, we will discuss the regulation of gene silencing across species, with special emphasis on the exosome's contribution to the process. We will also discuss the links between transcriptional and posttranscriptional mechanisms for gene silencing and their impact on the regulation of eukaryotic transcriptomes.
Abstract: Gli3 is an essential mediator of Sonic Hedgehog (Shh) response, acting both as a transcriptional activator and repressor. Mutations of Gli3 are also responsible for a number of developmental defects characterised by mental retardation and skeletal abnormalities. In the early chick embryo, Gli3 is mainly expressed in the CNS, limb and paraxial mesoderm. Initially expressed in a widespread manner in these tissues, Gli3 mRNA become restricted by Shh from the notochord in the case of the neural tube and mesoderm, and from the zone of polarizing activity (ZPA) in the limb bud. Thus, a gradient of Gli3 expression is observed in relation to the Shh source. Wnt and BMP pathways have also been implicated in Gli3 regulation. However, the mechanisms responsible for orchestrating Gli3 expression have not been investigated at the transcriptional level.
Here, I present a detailed analysis of the regulatory elements controlling Gli3 expression in the developing neural tube. In-silico analysis was used to determine the location of conserved non-coding elements, previously shown to contain enhancer modules. Eighteen non-coding elements were identified that are likely to regulate Gli3 expression. 5’ RACE demonstrated that in mammals all putative enhancer elements are intragenic, owing to the identification of a novel untranslated exon upstream of the annotated transcript. The activity of each element was tested in vivo using chick neural tube electroporation. One element was of particular interest, since it drives reporter gene expression in a manner that mimics that of endogenous Gli3. Further analysis of this region established that it contains binding sites for, and is regulated by TALE family transcription factors. These findings implicate for the first time TALE family proteins in the regulation of Gli3 expression, and offer a mechanism for integrating Shh and BMP signaling in the regulation of this developmentally important gene.
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