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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - RivRNAStructureDecay (Investigating the role of in vivo RNA structure in RNA degradation)

Teaser

Summary

RNA plays a central role in the regulation of gene expression. The basal levels of RNA in a cell depend on the ratio between RNA synthesis and RNA degradation. 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 study will be the first in vivo study on the role of RNA structures in RNA degradation and will provide the academic community with a completely novel mechanism on how to regulate RNA degradation to control protein production. This research is a multidisciplinary project that integrates molecular biology, nucleic acid chemistry and bioinformatics. Thus this research will broadly benefit these respective research fields.
This project was established to address three objectives:
Objective 1. Globally investigate RNA structural features and compare these to RNA degradation in vivo.
Objective 2. Decipher the mechanism of no go decay (NGD) via identifying the role of the G-quadruplex.
Objective 3. Determine the role of RNA structure in the miRNA pathway for both miRNA precursor processing and miRNA-directed processing.

Work performed

Objective 1. Globally investigate RNA structural features and compare these to RNA degradation in vivo.
We have developed the new genome-wide RNA structural-profiling platform SHAPE-Structure-seq using SHAPE-based chemical modification that is able to probe four nucleotides. We validated the platform by comparing phylogeny-derived rRNAs structure. Furthermore, we improved our new methodology and generated Cap-SHAPE-Structure-seq (CapS-Structure-seq) that enabled us to capture the RNA structure profiling of steady state mRNAs. This library only selects the full-length mature mRNAs to eliminate the degraded mRNAs. Cap-SHAPE-Structure-seq reveals clear signals on steady-state mRNAs. By analysing our RNA decay rate data generated from previous studies, we found the single-stranded region has a strong correlation with the decay rate. We identified the RNA structural motifs in steady state mRNAs, responsive to the RNA degradtion.

Objective 2. Decipher the mechanism of no go decay (NGD) via identifying the role of the G-quadruplex.
We identified the existence of RNA G-quadruplex in plants by using our new in vivo SHAPE-Structure-seq library and in vitro RNA G-quadruplex sequencing (rG4-seq). We are determining the role of RNA G-quadruplex in both RNA translation and degradation and our preliminary data suggests that RNA G-quadruplex may contribute to varying temperature response in plants.

Objective 3. Determine the role of RNA structure in the miRNA pathway for both miRNA precursor processing and miRNA-directed processing.
Using our in vivo Nuclear RNA SHAPE-Structure-seq (NRS-seq) platform we identified the RNA structural features in both pre-mRNAs and pri-miRNAs. We elucidated the role of RNA structural motifs in determining RNA splicing and validated our findings using transient assays together with corresponding individual RNA structural validations. We also found RNA structural features in pri-mRNAs that may link to pri-miRNA processing. This work is currently undergoing validation. Using our Cap-SHAPE-Structure-seq platform we identified the RNA structural motif on the steady state mRNAs required for miRNA-mediated degradation.

Final results

In the above section, we report multiple methodology developments which have advanced our understanding of the role of RNA structure in RNA degradation beyond the state-of-the-art. These developments represent exciting innovations to our existing genome-wide in vivo RNA structure profiling and individual RNA structure probing methods and have enabled the elucidation of the role of RNA structure in RNA splicing and miRNA-mediated degradation. In addition we have discoveried the existence of RNA G-quadruplex structural motifs in plants. We are progressing our aim to determine the role of RNA structure in different RNA degradation pathways. We are progressing our validation of the role of RNA structural motifs in pri-miRNA processing.
By the end of the project we anticipate that we will have successfully completed our obejctives and:
1. Determined whether the RNA structural motifs of RNA degradation through different RNA degradation pathways or all.
2. Determined the RNA structural motifs in pri-miRNA processing efficiency.
3. Developed other novel RNA structure profiling or probing methods to improve upon current methods.
4. Quantitatively identified in vivo how much RNA structural motifs contribute to RNA degradation.