TY - JOUR A1 - Berry, Scott A1 - Rosa, Stefanie A1 - Howard, Martin A1 - Buhler, Marc A1 - Dean, Caroline T1 - Disruption of an RNA-binding hinge region abolishes LHP1-mediated epigenetic repression JF - Genes & Development N2 - Epigenetic maintenance of gene repression is essential for development. Polycomb complexes are central to this memory, but many aspects of the underlying mechanism remain unclear. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binds Polycomb-deposited H3K27me3 and is required for repression of many Polycomb target genes in Arabidopsis. Here we show that LHP1 binds RNA in vitro through the intrinsically disordered hinge region. By independently perturbing the RNA-binding hinge region and H3K27me3 (trimethylation of histone H3 at Lys27) recognition, we found that both facilitate LHP1 localization and H3K27me3 maintenance. Disruption of the RNAbinding hinge region also prevented formation of subnuclear foci, structures potentially important for epigenetic repression. KW - chromatin KW - epigenetics KW - plant biology KW - Polycomb KW - RNA Y1 - 2017 U6 - https://doi.org/10.1101/gad.305227.117 SN - 0890-9369 SN - 1549-5477 VL - 31 SP - 2115 EP - 2120 PB - Cold Spring Harbor Laboratory Press CY - Cold Spring Harbor, NY ER - TY - JOUR A1 - Lombardo, Veronica A. A1 - Otten, Cecile A1 - Abdelilah-Seyfried, Salim T1 - Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis JF - Journal of visualized experiments N2 - The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts. KW - Developmental Biology KW - Issue 95 KW - zebrafish KW - embryo KW - heart KW - dissection KW - RNA KW - RT-qPCR Y1 - 2015 U6 - https://doi.org/10.3791/52087 SN - 1940-087X IS - 95 PB - JoVE CY - Cambridge ER - TY - JOUR A1 - Schudoma, Christian A1 - Larhlimi, Abdelhalim A1 - Walther, Dirk T1 - The influence of the local sequence environment on RNA loop structures JF - RNA : a publication of the RNA Society N2 - RNA folding is assumed to be a hierarchical process. The secondary structure of an RNA molecule, signified by base-pairing and stacking interactions between the paired bases, is formed first. Subsequently, the RNA molecule adopts an energetically favorable three-dimensional conformation in the structural space determined mainly by the rotational degrees of freedom associated with the backbone of regions of unpaired nucleotides (loops). To what extent the backbone conformation of RNA loops also results from interactions within the local sequence context or rather follows global optimization constraints alone has not been addressed yet. Because the majority of base stacking interactions are exerted locally, a critical influence of local sequence on local structure appears plausible. Thus, local loop structure ought to be predictable, at least in part, from the local sequence context alone. To test this hypothesis, we used Random Forests on a nonredundant data set of unpaired nucleotides extracted from 97 X-ray structures from the Protein Data Bank (PDB) to predict discrete backbone angle conformations given by the discretized eta/theta-pseudo-torsional space. Predictions on balanced sets with four to six conformational classes using local sequence information yielded average accuracies of up to 55%, thus significantly better than expected by chance (17%-25%). Bases close to the central nucleotide appear to be most tightly linked to its conformation. Our results suggest that RNA loop structure does not only depend on long-range base-pairing interactions; instead, it appears that local sequence context exerts a significant influence on the formation of the local loop structure. KW - RNA KW - 3D structure KW - structure prediction KW - Random Forests KW - machine learning KW - backbone conformation Y1 - 2011 U6 - https://doi.org/10.1261/rna.2550211 SN - 1355-8382 VL - 17 IS - 7 SP - 1247 EP - 1257 PB - Cold Spring Harbor Laboratory Press CY - Cold Spring Harbor, NY ER -