Stable m7G Cap-Distal 5'UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics

The 5' untranslated region (UTR) of diverse mRNAs contains secondary structures that can influence protein synthesis by modulating the initiation step of translation. Studies support the ability of these structures to inhibit 40S subunit recruitment and scanning, but the dynamics of ribosomal subunit interactions with mRNA remain poorly understood. Here, we developed a reconstituted Saccharomyces cerevisiae cell-free translation system with fluorescently labeled ribosomal subunits. We applied this extract and single-molecule fluorescence microscopy to monitor, in real time, individual 40S and 60S interactions with mRNAs containing 5' UTR hairpin structures with varying thermostability. In comparison to mRNAs containing no or weak 5' UTR hairpins (ΔG >= -5.4 kcal/mol), mRNAs with stable hairpins (ΔG <= -16.5 kcal/mol) showed reduced numbers of 60S recruitment to mRNA, consistent with the expectation of reduced translation efficiency for such mRNAs. Interestingly, such mRNAs showed increased numbers of 40S recruitment events to individual mRNAs but with shortened duration on mRNA. Correlation analysis showed that these unstable 40S binding events were nonproductive for 60S recruitment. Furthermore, although the mRNA sequence is long enough to accommodate multiple 40S, individual mRNAs are predominantly observed to engage with a single 40S at a time, indicating the sequestering of mRNA 5' end by initiating 40S. Altogether, these observations suggest that stable cap-distal hairpins in 5' UTR reduce initiation and translation efficiency by destabilizing 40S-mRNA interactions and promoting 40S dissociation from mRNA. The premature 40S dissociation frees mRNA 5'-end accessibility for new initiation events, but the increased rate of 40S recruitment is insufficient to compensate for the reduction of initiation efficiency due to premature 40S dissociation. This study provides the first single-molecule kinetic characterization of 40S/60S interactions with mRNA during cap-dependent initiation and the modulation of such interactions by cap-distal 5' UTR hairpin structures.

Molecular Dynamics Simulations of hydrophobic peptides that form β-hairpin structures in solution

Peptides designed with residues that have high propensity to occur in β-turns, form β-hairpin structures in apolar solvents as well in polar organic solvents such as dimethyl sulfoxide (DMSO), methanol and varying percentages of DMSO in chloroform (CHCl3). Presumably due to limited solubility, their conformations have not been investigated by experimental methods in water. We have examined the conformations of such designed peptides that fold into well-defined β-hairpin structures facilitated by β-turns, in the crystalline state and in solution, by Molecular Dynamics Simulations (MDS). The peptides fold into β-hairpin structures in water, starting from extended conformation. In DMSO, folding into β-hairpin structures was not observed, starting from extended conformation. However, when the starting structure is in β-hairpin conformation, unfolding is not observed during MDS in DMSO. Water clearly favours folding of short, hydrophobic peptides into β-turn and β-hairpin conformations from extended structures. DMSO does not have a denaturing effect on short, hydrophobic peptides.

The mitochondrial genome of Faughnia haani (Stomatopoda): novel organization of the control region and phylogenetic position of the superfamily Parasquilloidea

Background Stomatopod crustaceans are aggressive marine predators featuring complex compound eyes and powerful raptorial appendages used for “smashing” or “spearing” prey and/or competitors. Among them, parasquilloids (superfamily Parasquilloidea) possess eyes with 2-3 midband rows of hexagonal ommatidia and spearing appendages. Here, we assembled and analyzed the complete mitochondrial genome of the parasquilloid Faughnia haani and explored family- and superfamily-level phylogenetic relationships within the Stomatopoda based on mitochondrial protein coding genes (PCGs). Results The mitochondrial genome of F. haani is 16,089 bp in length and encodes 13 protein coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region that is relatively well organized, containing 2 GA-blocks, 4 poly-T stretches, various [TA(A)]n-blocks, and 2 hairpin structures. This organized control region is likely a synapomorphic characteristic in the Stomatopoda. Comparison of the control region among superfamilies shows that parasquilloid species are more similar to gonodactyloids than to squilloids and lysiosquilloids given the presence of various poly-T stretches between the hairpin structures and [TA(A)]n-blocks. Synteny is identical to that reported for other stomatopods and corresponds to the Pancrustacea ground pattern. A maximum-likelihood phylogenetic tree based on PCGs revealed that Parasquilloidea is sister to Lysiosquilloidea and Gonodactyloidea and not to Squilloidea, contradicting previous phylogenetic studies. Conclusions The novel phylogenetic position of Parasquilloidea revealed by our study indicates that ‘spearing’ raptorial appendages are plesiomorphic and that the ‘smashing’ type is either derived (as reported in previous studies) or apomorphic. Our results raise the possibility that the spearing raptorial claw may have independently evolved twice. The superfamily Parasquilloidea exhibits a closer relationship with other stomatopod superfamilies with a different raptorial claw type and with dissimilar numbers of midband rows of hexagonal ommatidia. Additional studies focusing on the assembly of mitochondrial genomes from species belonging to different genera, families, and superfamilies within the order Stomatopoda are warranted to reach a robust conclusion regarding the evolutionary history of this iconic clade based on mitochondrial PCGs.

Structure and unzipping behavior of dumbbell and hairpin DNA revealed by real-time nanopore sensing

Hairpin structures play an essential role in replication, transcription and recombination. Single molecule studies enable real-time measurement and observation of energetics and dynamics of hairpin structures, including folding and DNA-protein...

Sequence- and structure-selective mRNA m5C methylation by NSUN6 in animals

mRNA m5C, which has recently been implicated in the regulation of mRNA mobility, metabolism, and translation, plays important regulatory roles in various biological events. Two types of m5C sites are found in mRNAs. Type I m5C sites, which contain a downstream G-rich triplet motif and are computationally predicted to locate in the 5’ end of putative hairpin structures, are methylated by NSUN2. Type II m5C sites contain a downstream UCCA motif and are computationally predicted to locate in the loops of putative hairpin structures. However, their biogenesis remains unknown. Here we identified NSUN6, a methyltransferase that is known to methylate C72 of tRNAThr and tRNACys, as an mRNA methyltransferase that targets Type II m5C sites. Combining the RNA secondary structure prediction, miCLIP, and results from a high-throughput mutagenesis analysis, we determined the RNA sequence and structural features governing the specificity of NSUN6-mediated mRNA methylation. Integrating these features into an NSUN6-RNA structural model, we identified an NSUN6 variant that largely loses tRNA methylation but retains mRNA methylation ability. Finally, we revealed a weak negative correlation between m5C methylation and translation efficiency. Our findings uncover that mRNA m5C is tightly controlled by an elaborate two-enzyme system, and the protein-RNA structure analysis strategy established may be applied to other RNA modification writers to distinguish the functions of different RNA substrates of a writer protein.

A systematic analysis of the beta hairpin motif in the Protein Data Bank

The beta hairpin motif is a ubiquitous protein structural motif that can be found in molecules across the tree of life. This motif, which is also popular in synthetically designed proteins and peptides, is known for its stability and adaptability to broad functions. Here we systematically probe all 49,000 unique beta hairpin substructures contained within the Protein Data Bank (PDB) to uncover key characteristics correlated with stable beta hairpin structure, including amino acid biases and enriched inter-strand contacts. We also establish a set of broad design principles that can be applied to the generation of libraries encoding proteins or peptides containing beta hairpin structures.ImportanceThe beta hairpin motif is a common protein structural motif that is known for its stability and varied activity in diverse proteins. Here we use nearly fifty thousand beta hairpin substructures from the Protein Data Bank to systematically analyze and identify key characteristics of the beta hairpin motif. Ultimately, we provide a set of design principles for the generation of synthetic libraries encoding proteins containing beta hairpin structures.

Sequence- and structure-selective mRNA m5C methylation by NSUN6 in animals

AbstractmRNA m5C, which has recently been implicated in the regulation of mRNA mobility, metabolism, and translation, plays important regulatory roles in various biological events. Two types of m5C sites are found in mRNAs. Type I m5C sites, which contain a downstream G-rich triplet motif and are computationally predicted to locate in the 5’ end of putative hairpin structures, are methylated by NSUN2. Type II m5C sites contain a downstream UCCA motif and are computationally predicted to locate in the loops of putative hairpin structures. However, their biogenesis remains unknown. Here we identified NSUN6, a methyltransferase that is known to methylate C72 of tRNAThr and tRNACys, as an mRNA methyltransferase that targets Type II m5C sites. Combining the RNA secondary structure prediction, miCLIP, and results from a high-throughput mutagenesis analysis, we determined the RNA sequence and structural features governing the specificity of NSUN6-mediated mRNA methylation. Integrating these features into an NSUN6-RNA structural model, we identified an NSUN6 variant that largely loses tRNA methylation but retains mRNA methylation ability. Finally, we revealed a negative correlation between m5C methylation and translation efficiency. Our findings uncover that mRNA m5C is tightly controlled by an elaborate two-enzyme system, and the protein-RNA structure analysis strategy established may be applied to other RNA modification writers to distinguish the functions of different RNA substrates of a writer protein.

Vortical structures in the near wake of tabs with various geometries

The vortical structures and turbulence statistics in the near wake of rectangular, trapezoidal, triangular and ellipsoidal tabs were experimentally studied in a refractive-index-matching channel. The tabs share the same bulk dimensions, including a 17 mm height, a 28 mm base width and a $24.5^{\circ }$ inclination angle. Measurements were performed at two Reynolds numbers based on the tab height, $Re_{h}\simeq 2000$ (laminar incoming flow) and 13 000 (turbulent incoming flow). Three-dimensional, three-component particle image velocimetry (PIV) was used to study the mean flow distribution and dominant large-scale vortices, while complementary high-spatial-resolution planar PIV measurements were used to quantify high-order statistics. Instantaneous three-dimensional fields revealed the coexistence of a coherent counter-rotating vortex pair (CVP) and hairpin structures. The CVP and hairpin vortices (the primary structures) exhibit distinctive characteristics and strength across $Re_{h}$ and tab geometries. The CVP is coherently present in the mean flow field and grows in strength over a significantly longer distance at the low $Re_{h}$ due to the lower turbulence levels and the delayed shedding of the hairpin vortices. These features at the low $Re_{h}$ are associated with the presence of Kelvin–Helmholtz instability that develops over three tab heights downstream of the trailing edge. Moreover, a secondary CVP with an opposite sense of rotation resides below the primary one for the four tabs at the low $Re_{h}$. The interaction between the hairpin structures and the primary CVP is experimentally measured in three dimensions and shows complex coexistence. Although the CVP undergoes deformation and splitting at times, it maintains its presence and leads to significant mean spanwise and wall-normal flows.