Long noncoding RNAs and human disease

In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

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Landscape and variation of RNA secondary structure across the human transcriptome

RNA structure is crucial for gene regulation and function. In the past, transcriptomes have largely been parsed by primary sequences and expression levels, but it is now becoming feasible to annotate and compare transcriptomes based on RNA structure. In addition to computational prediction methods, the recent advent of experimental techniques to probe RNA structure by high-throughput sequencing has enabled genome-wide measurements of RNA structure and has provided the first picture of the structural organization of a eukaryotic transcriptome - the 'RNA structurome'. With additional advances in method refinement and interpretation, structural views of the transcriptome should help to identify and validate regulatory RNA motifs that are involved in diverse cellular processes and thereby increase understanding of RNA function.

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Understanding the transcriptome through RNA structure

Despite advances in biology and therapeutic modalities, existence of highly tumorigenic glioma stem-like cells (GSCs) makes glioblastomas (GBMs) invincible. N6-methyl adenosine (m6A), one of the abundant mRNA modifications catalyzed by methyltransferase-like 3 and 14 (METTL3/14), influences various events in RNA metabolism. Here, we report the crucial role of METTL3-mediated m6A modification in GSC (neurosphere) maintenance and dedifferentiation of glioma cells. METTL3 expression is elevated in GSC and attenuated during differentiation. RNA immunoprecipitation studies identified SOX2 as a bonafide m6A target of METTL3 and the m6A modification of SOX2 mRNA by METTL3 enhanced its stability. The exogenous overexpression of 3'UTR-less SOX2 significantly alleviated the inhibition of neurosphere formation observed in METTL3 silenced GSCs. METTL3 binding and m6A modification in vivo required intact three METTL3/m6A sites present in the SOX2-3'UTR. Further, we found that the recruitment of Human antigen R (HuR) to m6A-modified RNA is essential for SOX2 mRNA stabilization by METTL3. In addition, we found a preferential binding by HuR to the m6A-modified transcripts globally. METTL3 silenced GSCs showed enhanced sensitivity to γ-irradiation and reduced DNA repair as evidenced from the accumulation of γ-H2AX. Exogenous overexpression of 3'UTR-less SOX2 in METTL3 silenced GSCs showed efficient DNA repair and also resulted in the significant rescue of neurosphere formation from METTL3 silencing induced radiosensitivity. Silencing METTL3 inhibited RasV12 mediated transformation of mouse immortalized astrocytes. GBM tumors have elevated levels of METTL3 transcripts and silencing METTL3 in U87/TIC inhibited tumor growth in an intracranial orthotopic mouse model with prolonged mice survival. METTL3 transcript levels predicted poor survival in GBMs which are enriched for GSC-specific signature. Thus our study reports the importance of m6A modification in GSCs and uncovers METTL3 as a potential molecular target in GBM therapy.

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Essential role of METTL3-mediated m(6)A modification in glioma stem-like cells maintenance and radioresistance

A comprehensive understanding of RNA structure will provide fundamental insights into the cellular function of both coding and non-coding RNAs. Although many RNA structures have been analysed by traditional biophysical and biochemical methods, the low-throughput nature of these approaches has prevented investigation of the vast majority of cellular transcripts. Triggered by advances in sequencing technology, genome-wide approaches for probing the transcriptome are beginning to reveal how RNA structure affects each step of protein expression and RNA stability. In this Review, we discuss the emerging relationships between RNA structure and the regulation of gene expression.

Insights into RNA structure and function from genome-wide studies

Genome-wide association studies (GWAS) often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs) from the Human Gene Mutation Database (HGMD) that map to the untranslated regions (UTRs) of a gene. Rather than using minimum free energy approaches (e.g. mFold), we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, β-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD), and Hypertension), we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5′ UTRs of FTL and RB1) SNP–induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a “RiboSNitch,” that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

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Sam Broadaway

Papers

Disease-Associated Mutations That Alter the RNA Structural Ensemble