Li, Fan
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Publication Global Analysis of RNA Secondary Structure in Two Metazoans(2012-01-26) Li, Fan; Zheng, Qi; Ryvkin, Paul; Valladares, Otto; Murray, John I; Dragomir, Isabelle; Desai, Yaanik; Aiyer, Subhadra; Cherry, Sara; Wang, Li-San; Yang, Jamie; Gregory, Brian D; Bambina, Shelley; Sabin, Leah R; Lamitina, Todd; Rai, ArjunThe secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA]) and unpaired (single-stranded RNA [ssRNA]) components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.Publication SAVoR: A Server for Sequencing Annotation and Visualization of RNA Structures(2012-07-01) Li, Fan; Ryvkin, Paul; Childress, Daniel M; Valladares, Otto; Gregory, Brian D; Wang, Li-SanRNA secondary structure is required for the proper regulation of the cellular transcriptome. This is because the functionality, processing, localization and stability of RNAs are all dependent on the folding of these molecules into intricate structures through specific base pairing interactions encoded in their primary nucleotide sequences. Thus, as the number of RNA sequencing (RNA-seq) data sets and the variety of protocols for this technology grow rapidly, it is becoming increasingly pertinent to develop tools that can analyze and visualize this sequence data in the context of RNA secondary structure. Here, we present Sequencing Annotation and Visualization of RNA structures (SAVoR), a web server, which seamlessly links RNA structure predictions with sequencing data and genomic annotations to produce highly informative and annotated models of RNA secondary structure. SAVoR accepts read alignment data from RNA-seq experiments and computes a series of per-base values such as read abundance and sequence variant frequency. These values can then be visualized on a customizable secondary structure model. SAVoR is freely available at http://tesla.pcbi.upenn.edu/savor.Publication Genome-Wide Double-Stranded RNA Sequencing Reveals the Functional Significance of Base-Paired RNAs in Arabidopsis(2010-09-30) Zheng, Qi; Ryvkin, Paul; Li, Fan; Valladares, Otto; Wang, Li-San; Gregory, Brian D; Dragomir, Isabelle; Yang, Jamie; Cao, KajiaThe functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation require formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we marry classical nuclease-based structure mapping techniques with high-throughput sequencing technology to interrogate all base-paired RNA in Arabidopsis thaliana and identify ∼200 new small (sm)RNA–producing substrates of RNA–DEPENDENT RNA POLYMERASE6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. Although our methodology reveals the pairing status of RNA molecules in the absence of cellular proteins, previous studies have demonstrated that structural information obtained for RNAs in solution accurately reflects their structure in ribonucleoprotein complexes. Furthermore, our identification of RNA–DEPENDENT RNA POLYMERASE6 substrates and conserved functional RNA domains within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs using this approach strongly suggests that RNA molecules are correctly folded into their secondary structure in solution. Overall, our findings highlight the importance of base-paired RNAs in eukaryotes and present an approach that should be widely applicable for the analysis of this key structural feature of RNA.Publication Regulatory Impact of RNA Secondary Structure across the Arabidopsis Transcriptome(2012-11-01) Li, Fan; Vandivier, Lee E; Gregory, Brian D; Willmann, Matthew R; Chen, YingThe secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. However, the global influence of this feature on plant gene expression is still largely unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of rRNA-depleted (RNA sequencing), small RNA, and ribosome-bound RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis thaliana. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing evidence that secondary structure is necessary for these RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We further reveal that RNA folding is significantly anticorrelated with overall transcript abundance, which is often due to the increased propensity of highly structured mRNAs to be degraded and/or processed into small RNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. These findings provide a global assessment of RNA folding and its significant regulatory effects in a plant transcriptome.Publication Genome-Wide Analysis of RNA Secondary Structure in Eukaryotes(2013-01-01) Li, FanThe secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. Over the past decades, myriad studies have revealed specific examples of structural elements that direct the expression and function of both protein-coding messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In this work, we develop and apply a novel high-throughput, sequencing-based, structure mapping approach to study RNA secondary structure in three eukaryotic organisms. First, we assess global patterns of secondary structure across protein-coding transcripts and identify a conserved mark of strongly reduced base pairing at transcription start and stop sites, which we hypothesize helps with ribosome recruitment and function. We also find empirical evidence for reduced base pairing within microRNA (miRNA) target sites, lending further support to the notion that even mRNAs have additional selective pressures outside of their protein coding sequence. Next, we integrate our structure mapping approaches with transcriptome-wide sequencing of ribosomal RNA-depleted (RNA-seq), small (smRNA-seq), and ribosome-bound (ribo-seq) RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in the model organism Arabidopsis thaliana. We find that secondary structure and mRNA abundance are strongly anti-correlated, which is likely due to the propensity for highly structured transcripts to be degraded and/or processed into smRNAs. Finally, we develop a likelihood model and Bayesian Markov chain Monte Carlo (MCMC) algorithm that utilizes the sequencing data from our structure mapping approaches to generate single-nucleotide resolution predictions of RNA secondary structure. We show that this likelihood framework resolves ambiguities that arise from the sequencing protocol and leads to significantly increased prediction accuracy. In total, our findings provide on a global scale both validation of existing hypotheses regarding RNA biology as well as new insights into the regulatory and functional consequences of RNA secondary structure. Furthermore, the development of a statistical approach to structure prediction from sequencing data offers the promise of true genome-wide determination of RNA secondary structure.