Philip C. Bevilacqua

In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features

RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5′ splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

The double-stranded-RNA-binding motif: interference and much more

RNA duplexes have been catapulted into the spotlight by the discovery of RNA interference and related phenomena. But double-stranded and highly structured RNAs have long been recognized as key players in cell processes ranging from RNA maturation and localization to the antiviral response in higher organisms. Penetrating insights into the metabolism and functions of such RNAs have come from the identification and study of proteins that contain the double-stranded-RNA-binding motif.

5'-Triphosphate-Dependent Activation of PKR by RNAs with Short Stem-Loops

Molecular patterns in pathogenic RNAs can be recognized by the innate immune system, and a component of this response is the interferon-induced enzyme RNA-activated protein kinase (PKR). The major activators of PKR have been proposed to be long double-stranded RNAs. We report that RNAs with very limited secondary structures activate PKR in a 5′-triphosphate–dependent fashion in vitro and in vivo. Activation of PKR by 5′-triphosphate RNA is independent of RIG-I and is enhanced by treatment with type 1 interferon (IFN-α). Surveillance of molecular features at the 5′ end of transcripts by PKR presents a means of allowing pathogenic RNA to be distinguished from self-RNA. The evidence presented here suggests that this form of RNA-based discrimination may be a critical step in mounting an early immune response.

Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR.

RNA catalysis through compartmentalization

RNA performs important cellular functions in contemporary life forms. Its ability to act both as a catalyst and a storage mechanism for genetic information is also an important part of the RNA world hypothesis. Compartmentalization within modern cells allows the local concentration of RNA to be controlled and it has been suggested that this was also important in early life forms. Here, we mimic intracellular compartmentalization and macromolecular crowding by partitioning RNA in an aqueous two-phase system (ATPS). We show that the concentration of RNA is enriched by up to 3,000-fold in the dextran-rich phase of a polyethylene glycol/dextran ATPS and demonstrate that this can lead to approximately 70-fold increase in the rate of ribozyme cleavage. This rate enhancement can be tuned by the relative volumes of the two phases in the ATPS. Our observations support the importance of compartmentalization in the attainment of function in an RNA World as well as in modern biology.

A Brilliant Disguise for Self RNA: 5'-end and Internal Modifications of Primary Transcripts Suppress Elements of Innate Immunity

Interferon inducible protein kinase PKR is a component of innate immunity and mediates antiviral actions by recognizing pathogen associated molecular patterns (PAMPs). A well-known activator of PKR is long dsRNA, which can be produced during viral replication. Our recent results indicate that PKR can also be activated by short stem-loop RNA in a 5’-triphosphate-dependent fashion. A 5’-triphosphate is present primarily in foreign RNAs such as viral and bacterial transcripts, while a non-activating 5’-cap or 5’-monophosphate is present in most cellular RNAs. Recent studies indicate that internal RNA modifications and non-Watson-Crick motifs also repress PKR activation, and do so in an RNA structure-specific fashion. Interestingly, self-RNAs have more nucleoside modifications than non-self RNAs. Internal and 5’-end RNA modifications have repressive effects on other innate immune sensors as well, including TLR3, TLR7, TLR8, and RIG-I, suggesting that nucleoside modifications suppress innate immunity on a wide scale. (144/150)

Regulation of innate immunity through RNA structure and the protein kinase PKR.

Molecular recognition of RNA structure is key to innate immunity. The protein kinase PKR differentiates self from non-self by recognition of molecular patterns in RNA. Certain biological RNAs induce autophosphorylation of PKR, activating it to phosphorylate eukaryotic initiation factor 2α (eIF2α), which leads to inhibition of translation. Additional biological RNAs inhibit PKR, while still others have no effect. The aim of this article is to develop a cohesive framework for understanding and predicting PKR function in the context of diverse RNA structure. We present effects of recently characterized viral and cellular RNAs on regulation of PKR, as well as siRNAs. A central conclusion is that assembly of accessible long double-stranded RNA (dsRNA) elements within biological RNAs plays a key role in regulation of PKR kinase. Strategies for forming such elements include RNA dimerization, formation of symmetrical helical defects, A-form dsRNA mimicry, and coaxial stacking of helices.

Determination of in vivo RNA structure in low-abundance transcripts

RNA structure plays important roles in diverse biological processes. However, the structures of all but the few most abundant RNAs are presently unknown in vivo. Here we introduce DMS/SHAPE-LMPCR to query the in vivo structures of low-abundance transcripts. DMS/SHAPE-LMPCR achieves attomole sensitivity, a 100,000-fold improvement over conventional methods. We probe the structure of low-abundance U12 small nuclear RNA (snRNA) in Arabidopsis thaliana and provide in vivo evidence supporting our derived phylogenetic structure. Interestingly, in contrast to mammalian U12 snRNAs, the loop of the SLIIb in U12 snRNA is variable among plant species, and DMS/SHAPE-LMPCR determines it to be unstructured. We reveal the effects of proteins on 25S rRNA, 5.8S rRNA and U12 snRNA structure, illustrating the critical importance of mapping RNA structure in vivo. Our universally applicable method opens the door to identifying and exploring the specific structure-function relationships of the multitude of low-abundance RNAs that prevail in living cells.

Bioreactor droplets from liposome-stabilized all-aqueous emulsions

Artificial bioreactors are desirable for in vitro biochemical studies and as protocells. A key challenge is maintaining a favourable internal environment while allowing substrate entry and product departure. We show that semipermeable, size-controlled bioreactors with aqueous, macromolecularly crowded interiors can be assembled by liposome stabilization of an all-aqueous emulsion. Dextran-rich aqueous droplets are dispersed in a continuous polyethylene glycol (PEG)-rich aqueous phase, with coalescence inhibited by adsorbed ~130-nm diameter liposomes. Fluorescence recovery after photobleaching and dynamic light scattering data indicate that the liposomes, which are PEGylated and negatively charged, remain intact at the interface for extended time. Inter-droplet repulsion provides electrostatic stabilization of the emulsion, with droplet coalescence prevented even for submonolayer interfacial coatings. RNA and DNA can enter and exit aqueous droplets by diffusion, with final concentrations dictated by partitioning. The capacity to serve as microscale bioreactors is established by demonstrating a ribozyme cleavage reaction within the liposome-coated droplets.

RNA dimerization promotes PKR dimerization and activation.

The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2alpha, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.