RNA, Action through Interactions

Abstract

As transcription of the human genome is quite pervasive, it is possible that many novel functions of the noncoding genome have yet to be identified. Often the noncoding genome's functions are carried out by their RNA transcripts, which may rely on their structures and/or extensive interactions with other molecules. Recent technology developments are transforming the fields of RNA biology from studying one RNA at a time to transcriptome-wide mapping of structures and interactions. Here, we highlight the recent advances in transcriptome-wide RNA interaction analysis. These technologies revealed surprising versatility of RNA to participate in diverse molecular systems. For example, tens of thousands of RNA-RNA interactions have been revealed in cultured cells as well as in mouse brain, including interactions between transposon-produced transcripts and mRNAs. In addition, most transcription start sites in the human genome are associated with noncoding RNA transcribed from other genomic loci. These recent discoveries expanded our understanding of RNAs' roles in chromatin organization, gene regulation, and intracellular signaling.

Figure 1.

Overview of sequencing-based technologies for mapping RNA structures, RNA-RNA interactions, and RNA-DNA interactions.

Figure 2.. Sequencing-based technologies for mapping RNA structures.

(A) Summary of enzyme-based and chemical-based RNA structure technologies (columns) and their application domains (rows). Selected technologies (underscored) are expanded in detail in panels B. (B) Major steps of selected technologies. In PARS, polyA-tailed RNA is selected and divided into two pools. One pool is treated with RNase S1 that cleaves single-stranded sequence, and the other pool is treated with RNase V1 that cuts at double-stranded regions. The produced RNA segments are subjected to random fragmentation and converted into a sequencing library. In icSHAPE, cells are treated with NAI-azide, allowing for attaching a biotin moiety through copper-free CLICK reactions. SHAPE-reacted RNA segments are enriched by streptavidin-biotin interaction, and are subsequently converted into a sequencing library. In SHAPE-MaP, RNA is treated with 1M7 and is reverse transcribed in a reaction mixture that induces mutation at SHAPE-reacted sites.

Figure 3.. Sequencing-based technologies for mapping RNA-RNA interactions.

(A) Summary of antibody-based methods that analyze interactions mediated by a specific protein (left column) and genome-wide methods without targeting any specific proteins (right column). Selected technologies (underscored) are expanded in panels B. (B) Major steps of selected technologies. In PARIS, double-stranded RNA regions are crosslinked by AMT and UV. RNA is purified and subjected to proximity ligation. The resulting RNA is ligated with a 3’ adaptor and converted into a sequencing library. SPLASH procedure is similar to PARIS, except that instead of AMT, biotinylated psoralen is used as the crosslinking reagent, which allows for enrichment of double-stranded regions. LIGR-Seq used a similar experimental strategy, with different choices of RNA purification, treatment and ligation steps. In MARIO, RNA-protein complexes are crosslinked by UV. RNA is randomly fragmented and ligated with a biotinylated linker sequence and then subjected to proximity ligation. The resulting RNA-linker-RNA chimeric sequences are purified by streptavidin-biotin interaction and converted into a sequencing library.

Figure 4.. Sequencing-based technologies for mapping RNA-DNA interactions.

(A) Summary of technologies for RNA-DNA interactions based on a specific RNA (left column) or any RNA (right column). Selected technologies (underscored) are expanded in detail in panels B-D. (B-D) Major steps of selected technologies. (B) In MARGI, protein-RNA-DNA complexes are crosslinked by formaldehyde. DNA is fragmented. RNA is ligated with the RNA-end of a biotinylated half-RNA-half-DNA linker, and the DNA-end of this linker is subsequently ligated to DNA through proximity ligation. The resulting chimeric RNA-DNA sequences are selected by streptavidin-biotin interactions and converted into a sequencing library. (C-D) The ChAR-Seq and GRID-seq procedures are similar to MARGI. The major difference is that many steps are conducted in intact nuclei, including restriction enzyme digestion, RNA-linker ligation, and proximity ligation.