Widespread Influence of 3'-End Structures on Mammalian mRNA Processing and Stability.

The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, we investigate the possibility that mRNA structures facilitate the 3'-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. We find that RNA structures are predicted to be more prevalent within these extended 3'-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism.

A Stress Response that Monitors and Regulates mRNA Structure Is Central to Cold Shock Adaptation.

Temperature influences the structural and functional properties of cellular components, necessitating stress responses to restore homeostasis following temperature shift. Whereas the circuitry controlling the heat shock response is well understood, that controlling the E. coli cold shock adaptation program is not. We found that during the growth arrest phase (acclimation) that follows shift to low temperature, protein synthesis increases, and open reading frame (ORF)-wide mRNA secondary structure decreases. To identify the regulatory system controlling this process, we screened for players required for increased translation. We identified a two-member mRNA surveillance system that enables recovery of translation during acclimation: RNase R assures appropriate mRNA degradation and the Csps dynamically adjust mRNA secondary structure to globally modulate protein expression level. An autoregulatory switch in which Csps tune their own expression to cellular demand enables dynamic control of global translation. The universality of Csps in bacteria suggests broad utilization of this control mechanism.

Genome-Wide Analysis of RNA Secondary Structure.

Single-stranded RNA molecules fold into extraordinarily complicated secondary and tertiary structures as a result of intramolecular base pairing. In vivo, these RNA structures are not static. Instead, they are remodeled in response to changes in the prevailing physicochemical environment of the cell and as a result of intermolecular base pairing and interactions with RNA-binding proteins. Remarkable technical advances now allow us to probe RNA secondary structure at single-nucleotide resolution and genome-wide, both in vitro and in vivo. These data sets provide new glimpses into the RNA universe. Analyses of RNA structuromes in HIV, yeast, Arabidopsis, and mammalian cells and tissues have revealed regulatory effects of RNA structure on messenger RNA (mRNA) polyadenylation, splicing, translation, and turnover. Application of new methods for genome-wide identification of mRNA modifications, particularly methylation and pseudouridylation, has shown that the RNA "epitranscriptome" both influences and is influenced by RNA structure. In this review, we describe newly developed genome-wide RNA structure-probing methods and synthesize the information emerging from their application.

Genome-Wide Approaches for RNA Structure Probing.

RNA molecules of all types fold into complex secondary and tertiary structures that are important for their function and regulation. Structural and catalytic RNAs such as ribosomal RNA (rRNA) and transfer RNA (tRNA) are central players in protein synthesis, and only function through their proper folding into intricate three-dimensional structures. Studies of messenger RNA (mRNA) regulation have also revealed that structural elements embedded within these RNA species are important for the proper regulation of their total level in the transcriptome. More recently, the discovery of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) has shed light on the importance of RNA structure to genome, transcriptome, and proteome regulation. Due to the relatively small number, high conservation, and importance of structural and catalytic RNAs to all life, much early work in RNA structure analysis mapped out a detailed view of these molecules. Computational and physical methods were used in concert with enzymatic and chemical structure probing to create high-resolution models of these fundamental biological molecules. However, the recent expansion in our knowledge of the importance of RNA structure to coding and regulatory RNAs has left the field in need of faster and scalable methods for high-throughput structural analysis. To address this, nuclease and chemical RNA structure probing methodologies have been adapted for genome-wide analysis. These methods have been deployed to globally characterize thousands of RNA structures in a single experiment. Here, we review these experimental methodologies for high-throughput RNA structure determination and discuss the insights gained from each approach.

Hfq mediates transcriptome-wide RNA structurome reprogramming under virulence-inducing conditions in a phytopathogen.

Although RNA structures play important roles in regulating gene expression, the mechanism and function of mRNA folding in plant bacterial pathogens remain elusive. Therefore, we perform dimethyl sulfate sequencing (DMS-seq) on the Pseudomonas syringae under nutrition-rich and -deficient conditions, revealing that the mRNA structure changes substantially in the minimal medium (MM) that tunes global translation efficiency (TE), thereby inducing virulence. This process is led by the increased expression of hfq, which is directly activated by transcription regulators RpoS and CysB. The co-occurrence of Hfq and RpoS in diverse bacteria and the deep conservation of Hfq Y25 is critical for RNA-mediated regulation and implicates the wider biological importance of mRNA structure and feedback loops in the control of global gene expression.

Comprehensive in virio structure probing analysis of the influenza A virus identifies functional RNA structures involved in viral genome replication.

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures of vRNA are known to be involved in the viral proliferation process. Comprehensive vRNA structures in vitro, in virio, and in cellulo have been analyzed. However, the resolution of the structure map can be improved by comparative analysis and statistical modeling. Construction of a more high-resolution and reliable RNA structure map can identify uncharacterized functional structure motifs on vRNA in virion. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-seq with a robust statistical analysis. Our high-resolution analysis identified a stem-loop structure at nucleotide positions 39 - 60 of segment 6 and further validated the structure at nucleotide positions 87 - 130 of segment 5 that was previously predicted to form a pseudoknot structure in silico. Notably, when the cells were infected with recombinant viruses which possess the mutations to disrupt the structure, the replication and packaging of the viral genome were drastically decreased. Our results provide comprehensive and high-resolution information on the influenza A virus genome structures in virion and evidence that the functional RNA structure motifs on the influenza A virus genome are associated with appropriate replication and packaging of the viral genome.

Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer.

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

DMS-MaPseq for Genome-Wide or Targeted RNA Structure Probing In Vitro and In Vivo.

DMS-MaPseq is a chemical probing method combined with high throughput sequencing used to study RNA structure. Here we present a flexible protocol for adherent and suspension mammalian cells to analyze RNA structure in vitro or in vivo. The protocol provides instruction on either a targeted sequencing of a lncRNA of interest or a transcriptome-wide approach that provides structural data on all expressed RNAs, including lncRNAs. This technique is particularly useful for comparing in vitro and in vivo structure of RNAs, determining how mutations and polymorphisms with phenotypic effects influence RNA structure and analyzing RNA structure across the entire transcriptome.

HiPR: High-throughput probabilistic RNA structure inference.

Recent high-throughput structure-sensitive genome-wide sequencing-based assays have enabled large-scale studies of RNA structure, and robust transcriptome-wide computational prediction of individual RNA structures across RNA classes from these assays has potential to further improve the prediction accuracy. Here, we describe HiPR, a novel method for RNA structure prediction at single-nucleotide resolution that combines high-throughput structure probing data (DMS-seq, DMS-MaPseq) with a novel probabilistic folding algorithm. On validation data spanning a variety of RNA classes, HiPR often increases accuracy for predicting RNA structures, giving researchers new tools to study RNA structure.

Operon mRNAs are organized into ORF-centric structures that predict translation efficiency.

Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF.