Genome-Wide Analyses of RNA Structure-Function Relationships in Vivo

RNAs fold into complex secondary and tertiary structures. Until recently, however, no methods were available to probe the structures of all the RNAs (i.e. 'genome-wide') in a living system. In 2014, the Assmann and Bevilacqua labs developed such a method: 'Structure-seq' (Ding et al., Nature, 2014; Ritchey et al., NAR, 2017). Structure-seq capitalizes on the ability of a membrane-permeant chemical, dimethyl sulphate, to methylate As and Cs in RNA that are not base-paired; accordingly, the presence of DMS reactivity indicates the absence of base pairing. Such methylation imposes a stop to the processivity of reverse transcriptase, and so can be read-out genome-wide by next-gen sequencing techniques. We illustrated the efficacy of our method in the model plant Arabidopsis. Many abiotic stresses, including temperature, salt, heavy metals, and cellular crowding (desiccation) are known to change RNA structure in the test tube. We are currently applying our Structure-Seq method to investigate how such abiotic stresses change RNA structure genome-wide in rice in vivo, and how such changes in structure affect mRNA processing (transcription, translation, splicing, stability, etc.) and global RNA function.

Functional Roles of RNA G-Quadruplexes

Certain guanine (G)-rich RNA (or DNA) sequences can fold into a specialized structural motif termed the G-quadruplex structure (GQS) in the presence of potassium (K+) or sodium ions (Na+). The consensus sequence for a GQS is GxLaGxLbGxLcGx, where x is greater than or equal to 2 nucleotides (nt) and loops (L) a, b, and c are ≥ 1 nucleotide. Melissa Mullen, a previous Chemistry Ph.D. student co-advised in the Bevilacqua and Assmann labs, bioinformatically identified candidate GQS genome-wide in the model plant Arabidopsis (Mullen et al., 2010) and showed that some GQS fold with steep positive cooperativity as K+ concentrations increase (Mullen et al., 2011), implicating these structures as potential 'on-off' switches in gene regulation, and as potential sensors of ion concentrations. We are currently applying a combination of chemical and molecular biology approaches to assess the roles of plant GQS in vivo.

G-quartet and G-quadruplex structures and topologies. (a) G-quartet structure, showing Hoogsteen-to-Watson-Crick face hydrogen bonds and the central dehydrated monovalent ion integral to formation and stabilization. Unimolecular (b) parallel and (c) antiparallel G-quadruplex topologies. Adapted from (1,6,10,11). Dark lines follow the nucleic acid strand, arrowheads denote strand directionality, and gray boxes denote quadruplexes. The examples drawn here are for sequences having three quartets (Mullen et al., 2010).

Roles of RNA-Binding Proteins in Plant Stress Responses

Some years ago, we identified in broad bean (Vicia faba) a protein kinase, 'ABA-actived protein kinase' (AAPK) that is activated by the plant hormone abscisic acid (ABA) and is expressed primarily in guard cells (Li and Assmann, 1996; Li and Assmann, 2000). Expression of a dominant negative form of this kinase in Vicia guard cells inhibits ABA-activation of anion channels and ABA-induction of stomatal closure (Li et al., 2000). The ortholog of this protein kinase in the model plant Arabidopsis is named 'Open Stomata 1' (OST1) or 'SRK2E'. OST1 is a central component in guard cell ABA signaling (Acharya et al., 2013).

Expression of a GFP-tagged mutant
AAPK prevents stomatal closure
(Li et. al, 2000)

In Vicia faba, VfAKIP1, an RNA binding protein, is phosphorylated by AAPK in response to ABA (Li et al., 2002). Thus activated, AKIP1 binds a dehydrin transcript (Li et al., 2002) and relocalizes from the nucleoplasm to nuclear speckles (Li et al., 2002; Ng et al., 2004). The Arabidopsis genome encodes approximately 200 proteins with predicted RNA-binding properties, including three proteins with high similarity to VfAKIP1: UBA2a, UBA2b, and UBA2c. Our goals are to elucidate the functions of this small family of Arabidopsis heterogenous nuclear ribonucleoproteins (hnRNPs), and their regulation by ABA and other stresses. We have shown that overexpression of the Arabidopsis UBA2 proteins accelerates senescence (Bove et al., 2008; Kim et al., 2008; Na et al., 2015). We are also collaborating with the laboratory of RNA biochemist, Prof. Philip Bevilacqua, to investigate the RNA-binding properties of the UBA2 proteins.