Michelle M. Meyer

Library analysis of SCHEMA-guided protein recombination

The computational algorithm SCHEMA was developed to estimate the disruption caused when amino acid residues that interact in the three‐dimensional structure of a protein are inherited from different parents upon recombination. To evaluate how well SCHEMA predicts disruption, we have shuffled the distantly‐related β‐lactamases PSE‐4 and TEM‐1 at 13 sites to create a library of 214 (16,384) chimeras and examined which ones retain lactamase function. Sequencing the genes from ampicillin‐selected clones revealed that the percentage of functional clones decreased exponentially with increasing calculated disruption (E = the number of residue–residue contacts that are broken upon recombination). We also found that chimeras with low E have a higher probability of maintaining lactamase function than chimeras with the same effective level of mutation but chosen at random from the library. Thus, the simple distance metric used by SCHEMA to identify interactions and compute E allows one to predict which chimera sequences are most likely to retain their function. This approach can be used to evaluate crossover sites for recombination and to create highly mosaic, folded chimeras.

Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis

Estimates of the total number of bacterial species indicate that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins and RNAs. Bioinformatics searches of genomic DNA from bacteria commonly identify new noncoding RNAs (ncRNAs) such as riboswitches. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, indicating that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.

Integrating chemical footprinting data into RNA secondary structure prediction.

Chemical and enzymatic footprinting experiments, such as shape (selective 2′-hydroxyl acylation analyzed by primer extension), yield important information about RNA secondary structure. Indeed, since the -hydroxyl is reactive at flexible (loop) regions, but unreactive at base-paired regions, shape yields quantitative data about which RNA nucleotides are base-paired. Recently, low error rates in secondary structure prediction have been reported for three RNAs of moderate size, by including base stacking pseudo-energy terms derived from shape data into the computation of minimum free energy secondary structure. Here, we describe a novel method, RNAsc (RNA soft constraints), which includes pseudo-energy terms for each nucleotide position, rather than only for base stacking positions. We prove that RNAsc is self-consistent, in the sense that the nucleotide-specific probabilities of being unpaired in the low energy Boltzmann ensemble always become more closely correlated with the input shape data after application of RNAsc. From this mathematical perspective, the secondary structure predicted by RNAsc should be ‘correct’, in as much as the shape data is ‘correct’. We benchmark RNAsc against the previously mentioned method for eight RNAs, for which both shape data and native structures are known, to find the same accuracy in 7 out of 8 cases, and an improvement of 25% in one case. Furthermore, we present what appears to be the first direct comparison of shape data and in-line probing data, by comparing yeast asp-tRNA shape data from the literature with data from in-line probing experiments we have recently performed. With respect to several criteria, we find that shape data appear to be more robust than in-line probing data, at least in the case of asp-tRNA.

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.

Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria

In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria.

Scribl: An HTML5 Canvas-based graphics library for visualizing genomic data over the web

MOTIVATION High-throughput biological research requires simultaneous visualization as well as analysis of genomic data, e.g. read alignments, variant calls and genomic annotations. Traditionally, such integrative analysis required desktop applications operating on locally stored data. Many current terabyte-size datasets generated by large public consortia projects, however, are already only feasibly stored at specialist genome analysis centers. As even small laboratories can afford very large datasets, local storage and analysis are becoming increasingly limiting, and it is likely that most such datasets will soon be stored remotely, e.g. in the cloud. These developments will require web-based tools that enable users to access, analyze and view vast remotely stored data with a level of sophistication and interactivity that approximates desktop applications. As rapidly dropping cost enables researchers to collect data intended to answer questions in very specialized contexts, developers must also provide software libraries that empower users to implement customized data analyses and data views for their particular application. Such specialized, yet lightweight, applications would empower scientists to better answer specific biological questions than possible with general-purpose genome browsers currently available. RESULTS Using recent advances in core web technologies (HTML5), we developed Scribl, a flexible genomic visualization library specifically targeting coordinate-based data such as genomic features, DNA sequence and genetic variants. Scribl simplifies the development of sophisticated web-based graphical tools that approach the dynamism and interactivity of desktop applications. AVAILABILITY AND IMPLEMENTATION Software is freely available online at http://chmille4.github.com/Scribl/ and is implemented in JavaScript with all modern browsers supported.

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50–52% identical to known higher plant cycloartenol synthases and 40–49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

RNA structures regulating ribosomal protein biosynthesis in bacilli

In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species.

Complete RNA inverse folding: computational design of functional hammerhead ribozymes

Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/.

Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase

Histone modifiers play essential roles in controlling transcription and organizing eukaryotic genomes into functional domains. Here, we show that Set1, the catalytic subunit of the highly conserved Set1C/COMPASS complex responsible for histone H3K4 methylation (H3K4me), behaves as a repressor of the transcriptome largely independent of Set1C and H3K4me in the fission yeast Schizosaccharomyces pombe. Intriguingly, while Set1 is enriched at highly expressed and repressed loci, Set1 binding levels do not generally correlate with the levels of transcription. We show that Set1 is recruited by the ATF/CREB homolog Atf1 to heterochromatic loci and promoters of stress-response genes. Moreover, we demonstrate that Set1 coordinates with the class II histone deacetylase Clr3 in heterochromatin assembly at prominent chromosomal landmarks and repression of the transcriptome that includes Tf2 retrotransposons, noncoding RNAs, and regulators of development and stress-responses. Our study delineates a molecular framework for elucidating the functional links between transcriptome control and chromatin organization. DOI: http://dx.doi.org/10.7554/eLife.04506.001