Ming C. Hammond

RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP

Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.

The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches

A novel family of riboswitches, called SAM-IV, is the fourth distinct set of mRNA elements to be reported that regulate gene expression via direct sensing of S-adenosylmethionine (SAM or AdoMet). SAM-IV riboswitches share conserved nucleotide positions with the previously described SAM-I riboswitches, despite rearranged structures and nucleotide positions with family-specific nucleotide identities. Sequence analysis and molecular recognition experiments suggest that SAM-I and SAM-IV riboswitches share similar ligand binding sites, but have different scaffolds. Our findings support the view that RNA has considerable structural versatility and reveal that riboswitches exploit this potential to expand the scope of RNA in genetic regulation.

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP

Significance Bacteria are hidden forces of nature. For example, Geobacter bacteria play important roles in geochemistry by reducing metals in the environment. Scientists also are exploring the application of these bacteria toward toxic metal remediation and as “living batteries” that can generate electricity from biowaste. However, there is limited understanding of the signaling pathways that regulate this extracellular metal-reducing activity. Here we have discovered that Geobacter sulfurreducens use riboswitch sensors for a signaling molecule called cAG to regulate this process, which is an unexpected finding because cAG was previously associated only with pathogenic bacteria. Furthermore, we have adapted the riboswitch to generate a fluorescent biosensor that can be used to visualize cAG signaling in live bacteria. Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3′-5′, 3′-5′ cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP

Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.

Challenges of ligand identification for riboswitch candidates.

Expanding DNA sequence databases and improving methods for comparative analysis are being exploited to identify numerous noncoding RNA elements including riboswitches. Ligands for many riboswitch classes usually can be inferred based on the genomic contexts of representative RNAs, and complex formation or genetic regulation subsequently demonstrated experimentally. However, there are several candidate riboswitches for which ligands have not been identified. In this report, we discuss three of the most compelling riboswitch candidates: the ykkC/ykkD, yybP/ykoY and pfl RNAs. Each of these RNAs is numerous, phylogenetically widespread, and carries features that are hallmarks of metabolite-binding riboswitches, such as a well-conserved aptamer-like structure and apparent interactions with gene regulation elements such as ribosome binding sites or intrinsic transcription termination stems. These RNAs likely represent only a small sampling of the challenging motifs that researchers will encounter as new noncoding RNAs are identified.

Evidence for Widespread Gene Control Function by the ydaO Riboswitch Candidate

Nearly all representatives of experimentally validated riboswitch classes in bacteria control the expression of genes for the transport or synthesis of key metabolic compounds. Recent findings have revealed that some riboswitches also regulate genes involved in physiological changes, virulence, and stress responses. Many novel RNA motifs are being identified by using bioinformatics algorithms that search for conserved sequence and structural features located in intergenic regions. Some of these RNAs are likely to function as riboswitches for metabolites or signaling compounds, and confirmation of this function would reveal the basis of the genetic control of new regulons. Herein we describe the analysis of the ydaO riboswitch candidate, which represents one of the most widespread candidates remaining to be validated. These RNAs are common in Gram-positive bacteria, and their genomic associations with diverse genes suggest that they sense a compound that signals broader physiological changes. We determined that the ydaO motif exhibits sequence- and structure-dependent gene control, and reporter assays indicate that its natural ligand is present even when cells are grown in defined media. A transposon-mediated knockout screen resulted in mutants with a dysregulated expression of genes controlled by the RNA motif. The mutations disrupt genes that drastically modulate energy-generating pathways, suggesting that the intracellular concentration of the ligand sensed by the ydaO motif is altered under these stress conditions.

Nutritional Control of Epigenetic Processes in Yeast and Human Cells

The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. The effect of nutritional deficiency on H3K79 methylation was less pronounced, but was exacerbated in S. cerevisiae carrying a hypomorphic allele of Dot1, the enzyme responsible for H3K79 methylation. This result suggested a hierarchy of epigenetic modifications in terms of their susceptibility to nutritional limitations. Folate deficiency caused changes in gene transcription that mirrored the effect of complete loss of H3K4 methylation. Histone methylation was also found to respond to nutritional deficiency in the fission yeast Schizosaccharomyces pombe and in human cells in culture.

A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with clear resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. As the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein–mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.

Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP)

Significance Cyclic di-GMP (cdiG) is an important bacterial signaling molecule because it regulates motility and affects surface colonization and biofilm formation. It has long been established that cdiG is made by GGDEF enzymes, which are named after five conserved amino acids in the catalytic domain. However, a major group of bacteria, proteobacteria, have a high abundance of these enzymes, which raises the possibility that some of these enzymes have alternate functions. This study details the discovery of GGDEF enzymes that can also make cyclic AMP-GMP (cAG), a recently identified signaling molecule that regulates surface sensing and attachment in pathogenic and environmental bacteria. This provides the first evidence to our knowledge that GGDEF enzymes can make alternative cyclic dinucleotides to cdiG and that cAG is more widespread in proteobacteria. Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

Microfluidic Screening of Electrophoretic Mobility Shifts Elucidates Riboswitch Binding Function

Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high-throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for benchtop assays (3.2 versus 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation, and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high-resolution separation performance allows quantitation of equilibrium dissociation constants (K(d)) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and K(d) are reported for both a known and a candidate SAM-I riboswitch with comparison to in-line probing assay results. The microfluidic mobility shift assay establishes a scalable format for the study of riboswitch-ligand binding that will advance the discovery and selection of novel riboswitches and the development of antibiotics to target bacterial riboswitches.