Global mapping of RNA homodimers in living cells

Abstract

RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding RNA dimerization has been hampered by the lack of systematic in vivo detection methods. Here, we show that CLASH, PARIS, and other RNA proximity ligation methods detect RNA homodimers transcriptome-wide as “overlapping” chimeric reads that contain more than one copy of the same sequence. Analyzing published proximity ligation data sets, we show that RNA:RNA homodimers mediated by direct base-pairing are rare across the human transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA, and a subset of tRNAs. Mutations in the homodimerization domain of U8 snoRNA impede dimerization in vitro and disrupt zebrafish development in vivo, suggesting an evolutionarily conserved role of this domain. Analysis of virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N gene in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.

Classification and clustering of RNA crosslink-ligation data reveal complex structures and homodimers

“…The discovery of human patient mutations that disrupt the dimers points to the functional significance of such interactions ( Labrune et al 1996 ; Jenkinson et al 2016 ; Iwama et al 2017 ; Zhang et al 2021 ). While this manuscript was in preparation, the Kudla group published a similar approach and confirmed our discovery of homodimers in the snRNAs and snoRNAs ( Gabryelska et al 2022 ).…”

Chemical RNA Cross-Linking: Mechanisms, Computational Analysis, and Biological Applications

“…Recent exhaustive classification results in 5 major read types (Figure 9b): continuous or nongapped reads that are due to failed cross-linking and/or ligation (cont), two-segment or single-gapped reads due to single ligation events either within one RNA (gap1) or between two RNA molecules (trans), multisegment (>2 segments) or multigapped (>1 gap) reads due to simultaneous multi-cross-link and multiligation events on several RNA fragments in spatial proximity (gapm), and overlapping fragments that come from RNA homodimers (homo). 60,116,117 Reads with a single gap (gap1 and trans) suggest a single constraint in the structure, either a double helix cross-linked by psoralens, or tertiary contact/proximity cross-linked by SHARC or TBIA and other types of cross-linkers (Figure 9c). Reads with more than one gap suggest more complex structures, such as consecutive stemloops, pseudoknots and triplexes, depending on the relative location of the two stem regions (Figure 9d).…”

“…Classification of these reads reveals the fragment arrangements and therefore the underlying structural conformations. Recent exhaustive classification results in 5 major read types (Figure b): continuous or nongapped reads that are due to failed cross-linking and/or ligation (cont), two-segment or single-gapped reads due to single ligation events either within one RNA (gap1) or between two RNA molecules (trans), multisegment (>2 segments) or multigapped (>1 gap) reads due to simultaneous multi-cross-link and multiligation events on several RNA fragments in spatial proximity (gapm), and overlapping fragments that come from RNA homodimers (homo). ,, …”

Chemical RNA Crosslinking: Mechanisms, Computational Analysis and Biological Applications

“…9b): continuous or nongapped reads that are due to failed crosslinking and/or ligation (cont), two-segment or single-gapped reads due to single ligation events either within one RNA (gap1) or between two RNA molecules (trans), multi-segment (>2 segments) or multi-gapped (>1 gap) reads due to simultaneous multi-crosslink and multi-ligation events on several RNA fragments in spatial proximity (gapm), and overlapping fragments that come from RNA homodimers (homo). 57,108,109 Reads with a single gap (gap1 and trans) suggest a single constraint in the structure, either a double helix crosslinked by psoralens, or tertiary contact / proximity crosslinked by SHARC or TBIA and other types of crosslinkers (Fig. 9c).…”