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Investigating the role of in vivo RNA structure in RNA degradation

Periodic Reporting for period 4 - RivRNAStructureDecay (Investigating the role of in vivo RNA structure in RNA degradation)

Reporting period: 2020-07-01 to 2021-12-31

RNA plays a central role in the regulation of gene expression. RNA degradation is an active and critical process that dictates RNA levels and in part controls the relative levels of gene expression. However, despite many years of research in this area, important and perplexing questions remain about RNA stability. Even when comparable RNA degradation processes are involved individual RNAs can have distinct decay rates and the mechanisms that control such distinctive rates are unknown. RNA structure is likely to be intrinsic to our understanding of the RNA features that govern stability, and until very recently, our ability to measure the true in vivo RNA structure has been incredibly limited. We are addressing the role of RNA structure in the regulation of RNA degradation to determine the RNA structural features that regulate degradation pathways. This research is a multidisciplinary project that integrates molecular biology, nucleic acid chemistry and bioinformatics.
In summary, we have successfully developed five novel chemical profiling methods: SHAPE-Structure-seq (Yang et al., Genome Biology, 2021), CAP-Structure-seq (Yang et al., Nucleic acids Research, 2020), Nuc-Structure-seq (Liu et al., Genome Biology, 2021), SHALiPE-seq (Yang et al., Genome Biology, 2020) and smStructure-seq (Yang et al., Under 2nd review in Nature.). These methods revealed the RNA structural landscapes, facilitating the identification of the RNA structure-mediated RNA degradation elements. Apart from the RNA secondary structure involved in RNA degradation (Yang et al., Nucleic acids Research, 2020; Liu et al., Genome Biology, 2021, Yu et al., Frontiers in Molecular Bioscience, 2022 and Zhang et al., in preparation), we determined the existence of RNA tertiary structure motif, RNA G-quadruplex (Yang et al., Genome Biology, 2021) and revealed that the novel function of RNA G-quadruplex in regulating RNA stability (Yang et al., bioRxiv, 2022). Additionally, we have successfully discovered the novel functional role of GQS in No Go Decay and found that the GQS-mediated NGD is important in regulating plant root cell identity (Zhang et al., Nucleic Acids Research, 2019 and Duncan et al., In preparation). Notably, we have applied deep learning methods in identify RNA structure elements associated with RNA degradation (Yu et al., Frontiers in Molecular Bioscience, 2022 and Zhang et al., in preparation). We have comprehensively determined the mecahnisms of RNA structure-dependent miRNA-mediated RNA degradation. Building on the outputs from this objective, we obtained our ERC Proof of Concept proposal (ref. 966855 “ultraRNAs”), in which we propose to use our “Structure” rules to design and test the translational potential for using artificial miRNAs in cleaving plant viral RNAs, in particular, the beet yellows virus (BYV) in sugar beet, which is harmful virus causing up to 50% crop loss in recent years. Notably, our fundamental work on RNA structure-dependent regulation of RNA degradation derived from this project has been successfully applied in RNA structure-guided antisense oligonucleotide antiviral therapy for SARS-CoV-2 virus (Lulla, V et al., Journal of Virology, 2021).
Objective 1. Globally investigate RNA structural features and compare these to RNA degradation in vivo.
We have developed five novel chemical profiling methods for capture in vivo RNA structure features: 1) SHAPE-Structure-seq (Yang et al., Genome Biology, 2021), 2) CAP-Structure-seq (Yang et al., Nucleic acids Research, 2020), 3) Nuc-Structure-seq (Liu et al., Genome Biology, 2021), 4) SHALiPE-seq (Yang et al., Genome Biology, 2020), and 5) smStructure-seq (Yang, et al., Under 2nd review in Nature.). Notably, we have filed an international patent “Single-molecule RNA structure profiling method”. We have successfully taken advantage of the deep learning method for predicting RNA structure elements involved in RNA degradation (Yu et al., Frontiers in Molecular Bioscience, 2022 and Zhang et al., In preparation). The deep learning method for identifying the RNA degradation-mediated structure motifs will be subjected for method patent application. We have also successfully revealed the RNA structure features in regulating RNA processing including splicing and polyadenylation (Liu et al., Genome Biology, 2021).

Objective 2. Decipher the mechanism of no go decay (NGD) via identifying the role of the G-quadruplex.
Our breakthrough discovery has, for the first time, answered a longstanding question about whether RNA G-quadruplex structures exist in living eukaryotic cells (Yang et al., Genome Biology, 2020). We found that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice. Mutation of We discovered that RNA G-quadruplex structures are capable of triggering the RNA phase separation in living cells (Zhang et al., Nucleic Acids Research, 2019). We further found that the functional role of this GQS in the no go decay and its important impact in regulating plant root cell identity (Duncan, S., et al., In preparation).

Objective 3. Determine the role of RNA structure in the miRNA pathway for both miRNA precursor processing and miRNA-directed processing.
We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. We revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes (Yang et al., Nucleic acids Research, 2020).
We have generated two webservers Foldatlas and G4altas for the community.
Overall, we have fully finished all the tasks proposed in our objectives. We developed advanced and novel RNA structure probing methods and discovered the mechanism of RNA structure-mediated RNA degradation. Due to the COVID19, we have regenerated the transgenic plants and crossed plants during the 4th project periods. However, the setbacks during the pandemics have delayed the publication of the five associated manuscripts that will be published by the end of 2022 as follows:
1) Duncan et al., RNA G-quadruplex-mediated no go Decay regulates plant root cell identity. In preparation.
2) Zhang et al., An Evolutionarily Conserved Secondary Structure regulates miRNA cleavage. In preparation.
3) Zhang et al., Deep learning of RNA structure motifs mediated in the RNA degradation. In preparation.
4) Yang et al., In vivo single-molecule RNA structure analysis reveals functionally important COOLAIR structural diversity. Under 2nd review in Nature.
5) Yang et al., RNA G-quadruplex structure contributes to cold adaptation in plants. bioRxiv (2022) Under the revision in Nature Communications.
Summary Figure (From Xu et al., Science China Life Sciences 2022). Schematic of some exampled novel