Systematic detection of tertiary structural modules in large RNAs and RNP interfaces by Tb-seq

Abstract

Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg²⁺), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb³⁺), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb³⁺ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.

Fig. 1. Developing a sequencing-based approach to detect Tb³⁺ cleavage sites.

a 3D structure of group II intron with insert showing localization of metal ion in region of negative electrostatic potential region Adapted from PDB “4E8M”. b Mechanism of Tb³⁺ mediated cleavage. c Denaturing electrophoresis of ³²P-labeled aI5γ RNA probed at the indicated TbCl₃ concentrations. Source data are provided as a Source Data file. N = 2. d Primer extension electrophoresis of corresponding cDNA products from reverse transcription. Source data are provided as a Source Data file. N = 2. e Tb-seq library preparation workflow. A Tb³⁺ cleaved RNA and an untreated RNA are reverse transcribed with MarathonRT and a gene-specific RT primer containing a 5’ adapter handle. Illumina adapter (purple). The resulting cDNAs are ligated to a random hexamer fused to an Illumina adapter (pink) and PCR amplified to incorporate Illumina multiplex handles. Stop sites are processed using the RTEvents counter script (see Methods).

Fig. 2. Tb-seq of O.i. intron detects long-range, evolutionarily conserved RNA-RNA interactions.

a Secondary structure of the O.i. intron displaying sites of strong Tb³⁺cleavage (red). Long-range RNA-RNA interactions are indicated by Greek letters. EBS1 and EBS2 correspond to exon binding sites. Gray nucleotides indicate a lack of sequencing data in this region. b 3-D structure of O.i. intron showing sites of strong Tb³⁺ cleavage on the RNA backbone (red highlight). Inserts showing close-up view of two long-range RNA-RNA interactions (ζ-ζ’ and λ-λ’) with nucleotides that display Tb³⁺ cleavage (red) and hydrogen bonds (purple dashed lines). Adapted from PDB “4E8M”. Source data are provided as a Source Data file.

Fig. 3. Tb-seq of HCV IRES detects conserved L-shaped bend in stem loop II.

a Secondary of HCV 5’ UTR Stem loop (SL) II displaying sites of strong Tb³⁺cleavage (red). b 3-D structure of SLII showing sites of strong Tb³⁺ cleavage on the RNA backbone (red highlight). Adapted from PDB “5A2Q”. Source data are provided as a Source Data file.

Fig. 4. Probing RNA-Protein interactions in human RNase P.

a 3-D structure of RNase P complexed with its protein components showing sites of strong Tb³⁺ cleavage on the RNA backbone (red highlight). Inserts show close up views of two regions containing RNA-Protein interactions with nucleotides that display Tb³⁺ cleavage labeled (red) and hydrogen bonds (purple dashed lines). Adapted from PDB “6AHR”. b Δ Tb reactivity profile for the first 150nt of RNase P. Nucleotides indicated in green become less reactive when probed in the absence of protein components, whereas nucleotides indicated in blue become more reactive. Source data are provided as a Source Data file.

Fig. 5. Tb-seq identifies structural modules in SARS-CoV-2.

Cell lysate probing of the 5’ terminal of SARS-COV2. Secondary structure of the SARS-CoV-2 displaying sites of strong Tb³⁺cleavage (red). Inserts showing close-up views of two regions that display Δ Tb reactivities. Source data are provided as a Source Data file.