Following the intracellular localization of the iab-8ncRNA of the bithorax complex using the MS2-MCP-GFP system

Abstract

Homeotic genes are aligned on the chromosome in the order of the segments that they specify along the antero-posterior axis of the fly. In general the genes affecting the more posterior segments repress the more anterior genes, a phenomenon known as "posterior dominance". There is however a noticeable exception to this rule in the central nervous system of Drosophila melanogaster where the posterior Abd-B gene does not repress the immediately more anterior abd-A gene. Instead, abd-A repression is accomplished by a 92 kb-long ncRNA (the iab-8ncRNA) that is transcribed from the large inter-genic region between abd-A and Abd-B. This iab-8ncRNA encodes a microRNA to repress abd-A and also a second redundant repression mechanism acting in cis and thought to be transcriptional interference with the abd-A promoter. Using in situ hybridization, a previous work suggested that the iab8ncRNA transcript forms discrete foci restricted to the nuclear periphery and that this localization may be important for its function. In order to better characterize the intra-cellular localization of the iab-8ncRNA we used the MS2-MCP system, which allows fluorescent labeling of RNA in cells and relies on the interaction between GFP-tagged MS2 coat protein (MCP-GFP) and MS2 RNA stem loops. Our results indicate that the large foci seen in previous studies correspond to the site of iab8ncRNA transcription and that the foci seen may simply be an indication of the level of transcription at the locus. We find no evidence to suggest that this localization is important for its function on abd-A repression. We discuss the idea that the iab-8ncRNA may be a relic of a more general ancient mechanism of posterior dominance during the emergence of the hox clusters that was mediated by transcriptional interference.

CTCF: making the right connections

“…If convergent orientation at contact sites were energetically preferred, it would overcome any of the relatively small costs of bending the large chromatin region within the loop and there would be no requirement that sites be oriented on the DNA sequence (but see the comment in legend of Fig. 5, below;Arib et al 2015) It is apparent that these domains are formed by a nonequilibrium process, and some recent studies indicate what form it might take. In a review of Guo and colleagues (Rao et al 2014;Guo et al 2015), Nichols and Corces (2015) suggested that the ability of CTCF to bend DNA at one end of its binding site (Arnold et al 1996; MacPherson and Sadowski 2010) would create an incipient loop, which could then enlarge until a mating CTCF was encountered.…”

CRISPR/Cas9 and FLP-FRT mediated regulatory dissection of the BX-C of Drosophila melanogaster

“…The effects of mutations in such genes can be probed to understand the nature of variability present in the system. The other possibility pertaining to the differential dosages of ncRNAs can be probed by in-situ hybridization (Arib et al 2015). However, the de nitive changes in the ncRNAs corresponding with the adult phenotypes require further investigation.…”

Fixed and live visualization of RNAs in Drosophila oocytes and embryos

“…We subsequently adapted this system for transgenic use in Drosophila in order to visualize nanos (nos) mRNA during oogenesis, and showed that localization occurred by diffusion/entrapment [26]. It has since been used by us and others to investigate trafficking of a variety of mRNAs in oocytes, embryos, and neurons [19, 27–31] (Figures 3, 4) and a recent study has illustrated its utility for investigating localization of long non-coding RNAs [32]. The transgenic MS2/MCP system has also been used in Drosophila embryos to monitor native transcripts and transcriptional dynamics in individual nuclei cells and to track transcriptional activity within groups of cells [33, 34].…”