Archaeal tRNA-Splicing Endonuclease as an Effector for RNA Recombination and Novel Trans-Splicing Pathways in Eukaryotes

We have characterized a homodimeric tRNA endonuclease from the euryarchaeota Ferroplasma acidarmanus (FERAC), a facultative anaerobe which can grow at temperatures ranging from 35 to 42 °C. This enzyme, contrary to the eukaryal tRNA endonucleases and the homotetrameric Methanocaldococcus jannaschii (METJA) homologs, is able to cleave minimal BHB (bulge–helix–bulge) substrates at 30 °C. The expression of this enzyme in Schizosaccharomyces pombe (SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts. In addition, the FERAC endonuclease can create proteins with new functionalities through the recombination of protein domains.

Nonenzymatic loop-closing ligation generates RNA hairpins and is a template-free way to assemble functional RNAs

RNA hairpin loops are the predominant element of secondary structure in functional RNAs. The emergence of primordial functional RNAs, such as ribozymes that fold into complex structures that contain multiple hairpin loops, is generally thought to have been supported by template-directed ligation. However, template inhibition and RNA misfolding problems impede the emergence of function. Here we demonstrate that RNA hairpin loops can be synthesized directly from short RNA duplexes with single-stranded overhangs by nonenzymatic loop-closing ligation chemistry. We show that loop-closing ligation allows full-length functional ribozymes containing a hairpin loop to be assembled free of inhibitory template strands. This approach to the assembly of structurally complex RNAs suggests a plausible pathway for the emergence of functional RNAs before a full-length RNA copying process became available.

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance.

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake, and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we have performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli. By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we have mapped the topology of the protein. Using these data, we have experimentally validated a structural model of YqjA generated using evolutionary co-variance, which consists of an a-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggests that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

FindNonCoding: rapid and simple detection of non-coding RNAs in genomes

Summary Non-coding RNAs are often neglected during genome annotation due to their difficulty of detection relative to protein coding genes. FindNonCoding takes a pattern mining approach to capture the essential sequence motifs and hairpin loops representing a non-coding RNA family and quickly identify matches in genomes. FindNonCoding was designed for ease of use and accurately finds non-coding RNAs with a low false discovery rate. Availability FindNonCoding is implemented within the DECIPHER package (v2.19.3) for R (v4.1) available from Bioconductor. Pre-trained models of common non-coding RNA families are included for bacteria, archaea, and eukarya. Supplementary information Supplementary data are available at Bioinformatics online.

The effect of hairpin loop on the structure and gene expression activity of the long-loop G-quadruplex

G-quadruplex (G4), a four-stranded DNA or RNA structure containing stacks of guanine tetrads, plays regulatory roles in many cellular functions. So far, conventional G4s containing loops of 1–7 nucleotides have been widely studied. Increasing experimental evidence suggests that unconventional G4s, such as G4s containing long loops (long-loop G4s), play a regulatory role in the genome by forming a stable structure. Other secondary structures such as hairpins in the loop might thus contribute to the stability of long-loop G4s. Therefore, investigation of the effect of the hairpin-loops on the structure and function of G4s is required. In this study, we performed a systematic biochemical investigation of model G4s containing long loops with various sizes and structures. We found that the long-loop G4s are less stable than conventional G4s, but their stability increased when the loop forms a hairpin (hairpin-G4). We also verified the biological significance of hairpin-G4s by showing that hairpin-G4s present in the genome also form stable G4s and regulate gene expression as confirmed by in cellulo reporter assays. This study contributes to expanding the scope and diversity of G4s, thus facilitating future studies on the role of G4s in the human genome.

Folds induced by multiple parallel or antiparallel double-helices: (pseudo)knotting of single-stranded RNA

ABSTRACTWe develop tools to explore and catalogue the topologies of knotted or pseudoknotted circular folds due to secondary and tertiary interactions within a closed loop of RNA which generate multiple double-helices due (for example) to strand complementarity. The fold topology is captured by a ‘contracted fold’ which merges helices separated by bulges and removes hairpin loops. Contracted folds are either trivial or pseudoknotted. Strand folding is characterised by a rigid-vertex ‘polarised strand graph’, whose vertices correspond to double-helices and edges correspond to strands joining those helices. Each vertex has a plumbline whose polarisation direction defines the helical axis. That polarised graph has a corresponding circular ribbon diagram and canonical alphanumeric fold label. Key features of the ‘fully-flagged’ fold are the arrangement of complementary domains along the strand, described by a numerical bare fold label, and a pair of binary ‘flags’: a parity flag that specifies the twist in each helix (even or odd half-twists), and an orientation flag that characterises each double-helix as parallel or antiparallel. A simple algorithm is presented to translate an arbitrary fold label into a polarised strand graph. Any embedding of the graph in 3-space is an admissible fold geometry; the simplest embeddings minimise the number of edge-crossings in a planar graph drawing. If that number is zero, the fold lies in one of two classes: (a)-type ‘relaxed’ folds, which contain conventional junctions and (b)-type folds whose junctions are described as meso-junctions in H. Wang and N.C. Seeman, Biochem, vol. 34, pp920-929. (c)-type folds induce polarised strand graphs with edge-crossings, regardless of the planar graph drawing. Canonical fold labelling allows us to sort and enumerate all ‘semi-flagged’ folds with up to six contracted double-helices as windings around the edges of a graph-like fold skeleton, whose cyclomatic number - the ‘fold genus’ - ranges from 0 – 3, resulting in a pair of duplexed strands along each skeletal edge. Those semi-flagged folds admit both even and odd double-helical twists. Appending specific parity flags to those semi-flagged folds gives fully-flagged (a)-type folds, which are also enumerated up to genus-3 cases. We focus on all-antiparallel folds, characteristic of conventional ssRNA and enumerate all distinct (a), (b) and (c)-type folds with up to five double-helices. Those circular folds lead to pseudoknotted folds for linear ssRNA strands. We describe all linear folds derived from (a) or (b)-type circular folds with up to four contracted double-helices, whose simplest cases correspond to so-called H, K and L pseudoknotted folds, detected in ssRNA. Fold knotting is explored in detail, via constructions of so-called antifolds and isomorphic folds. We also tabulate fold knottings for (a) and (b)-type folds whose embeddings minimise the number of edge-crossings and outline the procedure for (c)-type folds. The inverse construction - from a specific knot to a suitable nucleotide sequence - results in a hierarchy of knots. A number of specific alternating knots with up to 10 crossings emerge as favoured fold designs for ssRNA, since they are readily constructed as (a)-type all-antiparallel folds.

An extended APOBEC3A mutation signature in cancer

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

Single-Conformation Spectroscopy of Cold, Protonated DPG-containing peptides: Switching β-turn Types and Formation of a sequential Type II/II’ Double β-turn

D-proline (DPro, DP) is widely utilized to form β-hairpin loops in engineered peptides that would otherwise be unstructured, most often as part of a DPG sub-unit that forms a β-turn....

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

Metazoan pri-miRNAs and pre-miRNAs fold into characteristic hairpins that are recognized by the processing machinery. Essential to the recognition of these miR-precursors are their apical junctions where double-stranded stems meet single-stranded hairpin loops. Little is known about how apical junctions and loops fold in three-dimensional space. Here we developed a scaffold-directed crystallography method and determined the structures of eight human miR-precursor apical junctions and loops. Six structures contain non-canonical base pairs stacking on top of the hairpin stem. U-U pair contributes to thermodynamic stability in solution and is highly enriched at human miR-precursor apical junctions. Our systematic mutagenesis shows that U-U is among the most efficiently processed variants. The RNA-binding heme domain of pri-miRNA-processing protein DGCR8 binds longer loops more tightly and non-canonical pairs at the junction appear to modulate loop length. Our study provides structural and biochemical bases for understanding miR-precursors and molecular mechanisms of microRNA maturation.