Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

Long non-coding RNAs (lncRNAs) are increasingly appreciated for their important functions in mammalian cells. However, how their functional capacities are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA-binding proteins, but the structural principles that underlie this mode of regulation are unknown. The NORAD lncRNA is a known decoy for Pumilio proteins, which modulate the translation and stability of hundreds of mRNAs, and, consequently, a regulator of genomic stability and aging. We probed the RNA structure and long-range RNA-RNA interactions formed by human NORAD inside cells under different stressful conditions. We discovered a highly modular structure consisting of well-defined domains that contribute independently to NORAD function. Following arsenite stress, most structural domains undergo relaxation and form interactions with other RNAs that are targeted to stress granules. We further revealed a unique structural organization that spatially clusters the multiple Pumilio binding sites along NORAD and consequently contributes to the de-repression of Pumilio targets. We then applied these structural principles to design an effective artificial decoy for the let-7 miRNA. Our work demonstrates how the sequence of a lncRNA spatially clusters its function into separated domains and how structural principles can be employed for the rational design of lncRNAs with desired activities

Erwachsenenbildung im Kontext: theoretische Rahmungen, empirische Spielräume und praktische Regulative; Festschrift zum 60. Geburtstag von Jürgen Wittpoth

Ein Rahmen ist keine Grenze - er kann auch zur Auseinandersetzung mit dem gerahmten Inhalt und zur Rahmenüberschreitung anregen. Die Werke von Piet Mondrian gehören zu den bekanntesten "rahmensprengenden" Bildern. In der Erwachsenenbildung stehen die Arbeiten von Jürgen Wittpoth für rahmenüberschreitende Gedanken und Konzepte. Schon 1994 beschäftigt sich Jürgen Wittpoth in seiner Habilitationsschrift "Rahmungen und Spielräume des Selbst" kritisch mit den Grenzen, die empirische Befunde der Erwachsenenbildung setzen. In diesem Sammelband, der zum 60. Geburtstag von Jürgen Wittpoth erschienen ist, betrachten namhafte Wegbeleiter seine wegweisenden Arbeiten im Kontext von Theorie, Empirie und praktischen Regulativen

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian intitiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites

Autorepression of Rfx1 Gene Expression: Functional Conservation from Yeast to Humans in Response to DNA Replication Arrest

The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage