Abstract
We have previously developed and described a method for measuring RNA co-locations within cells, called Proximity RNA-seq, which promises insights into RNA expression, processing, storage and translation. Here, we describe transcriptome-wide proximity RNA-seq datasets obtained from human neuroblastoma SH-SY5Y cell nuclei. To aid future users of this method, we also describe and release our analysis pipeline, CloseCall, which maps cDNA to a custom transcript annotation and allocates cDNA-linked barcodes to barcode groups. CloseCall then performs Monte Carlo simulations on the data to identify pairs of transcripts, which are co-barcoded more frequently than expected by chance. Furthermore, derived co-barcoding frequencies for individual transcripts, dubbed valency, serve as proxies for RNA density or connectivity for that given transcript. We outline how this pipeline was applied to these sequencing datasets and openly share the processed data outputs and access to a virtual machine that runs CloseCall. The resulting data specify the spatial organization of RNAs and builds hypotheses for potential regulatory relationships between RNAs.
Highlights
Background & SummaryThe positioning of RNA molecules and the spatial organization of transcriptomes in cells is poorly understood to date
Nascent RNA emerges from its encoding gene during synthesis, and genome folding and gene positioning determines the spatial point of origin for any RNA molecule
This is achieved by chromatin retention through interactions between chromatin-bound proteins or the genome itself and the RNA molecule or by a very short half-life of the RNA and its prompt degradation in the close vicinity of the gene[2]
Summary
Background & SummaryThe positioning of RNA molecules and the spatial organization of transcriptomes in cells is poorly understood to date. Some transcripts reside within nuclei and are frequently localized in proximity of their encoding gene This is achieved by chromatin retention through interactions between chromatin-bound proteins or the genome itself and the RNA molecule or by a very short half-life of the RNA and its prompt degradation in the close vicinity of the gene[2]. Such spatial restraints can have implications for RNAs regulating the expression of other genes, by limiting the number of accessible target genes in 3-dimensional space to the immediate neighbourhood of the regulatory RNA. The property of RNA and proteins to separate from surroundings and to form bodies to compartmentalize cellular tasks can further specify RNA localization but becomes detrimental for cells when mutations in molecules resident to such structures are acquired that lead to irreversible aggregation and disease states, as observed for example for RNA repeats[9]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call