High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

ABSTRACTIn the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination.Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

Take Me Home, Protein Roads: Structural Insights into Signal Peptide Interactions during ER Translocation

Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins’ target location and provide translocation fidelity. In this review, we follow the signal peptide into the ER and discuss the recent insights that structural biology has provided on the governing principles of those interactions.

Co-dependency of exchanged behaviors is a cue for agency attribution in 10-month-olds

Goal-directed social interactions (whether instrumental or communicative) involve co-dependent, partially predictable actions of interacting agents as social goals cannot be achieved by continuously exchanging the same, perfectly predictable, or completely random behaviors. We investigated whether 10-month-olds are sensitive to the co-dependence and degree of predictability in an interactive context where unfamiliar entities exchanged either perfectly predictable (identical), partially predictable (co-dependent), or non-predictable (random) signal sequences. We found that when—following the interactive exchanges—one of the entities turned in the direction of one of two lateral target objects, infants looked more at the indicated referent, but only in the partially predictable signals condition. This shows that infants attributed agency to the orienting entity and interpreted its turning action as a referential object-directed action. The present findings suggest that the co-dependency and partial predictability of exchanged behaviors can serve as an abstract structural cue to attribute intentional agency and recognize goal-directed social interactions.

Bacterial signal peptides: structure, optimization, and applications

Bacterial signal peptides are N-terminal tags that direct proteins for export through one of various transport pathways. These signal peptides are highly important as they are the key determinants of transport, ensuring that the correct protein ends up at the correct pathway. While these peptides consist of three domains with well conserved biochemical properties, there still remains a large amount of diversity between the signal sequences for different proteins, transport pathways, and bacterial species. Recent advancements have allowed us to predict signal sequences and manipulate them in an attempt to optimize export efficiency. This knowledge can then be exploited in the field of recombinant protein production wherein bacterial species can be used to produce and secrete proteins of interest. By fusing the protein with an optimized signal peptide, the yield or rate of export can be improved. This review focuses on signal peptides for two primary transport pathways (Sec and Tat) in E. coli specifically, with an emphasis on applications and the production of recombinant proteins.

Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are hindered by mosquito and pathogen resistance, so research has turned to altering the microbiota of the vectors. In this strategy, called paratransgenesis, symbiotic bacteria are genetically modified to affect the mosquito’s phenotype by engineering them to deliver antiplasmodial effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. However, common secretion signals from E. coli and closely related species do not function in Asaia. Here, we report evaluation of 20 native Asaia N-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of antiplasmodial effector molecules directed to the periplasm and ultimately outside the cell. We tested the hypothesis that by increasing the amount of antiplasmodials released from the cell we would also increase parasite killing power. We scanned the Asaia bogorensis SF2.1 genome to identify signal sequences from extra-cytoplasmic proteins and fused these to the reporter protein alkaline phosphatase. Six signals resulted in significant levels of protein released from the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three Asaia strains were able to suppress the number of oocysts formed after a blood meal containing P. berghei to a significantly greater degree than wild-type Asaia, although prevalence was not decreased beyond levels obtained with a previously isolated siderophore receptor signal sequence. We interpret these results to indicate that there is a maximum level of suppression that can be achieved when the effectors are constitutively driven due to stress on the symbionts. This suggests that simply increasing the amount of antiplasmodial effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains and that symbiont fitness must be considered as well.

Differential Entropy Feature Signal Extraction Based on Activation Mode and Its Recognition in Convolutional Gated Recurrent Unit Network

In brain-computer-interface (BCI) devices, signal acquisition via reducing the electrode channels can reduce the computational complexity of models and filter out the irrelevant noise. Differential entropy (DE) plays an important role in emotional components of signals, which can reflect the area activity differences. Therefore, to extract distinctive feature signals and improve the recognition accuracy based on feature signals, a method of DE feature signal recognition based on a Convolutional Gated Recurrent Unit network was proposed in this paper. Firstly, the DE and power spectral density (PSD) of each original signal were mapped to two topographic maps, and the activated channels could be selected in activation modes. Secondly, according to the position of original electrodes, 1D feature signal sequences with four bands were reconstructed into a 3D feature signal matrix, and a radial basis function interpolation was used to fill in zero values. Then, the 3D feature signal matrices were fed into a 2D Convolutional Neural Network (2DCNN) for spatial feature extraction, and the 1D feature signal sequences were fed into a bidirectional Gated Recurrent Unit (BiGRU) network for temporal feature extraction. Finally, the spatial-temporal features were fused by a fully connected layer, and recognition experiments based on DE feature signals at the different time scales were carried out on a DEAP dataset. The experimental results showed that there were different activation modes at different time scales, and the reduction of the electrode channel could achieve a similar accuracy with all channels. The proposed method achieved 87.89% on arousal and 88.69% on valence.

Two Signal Recognition Particle Sequences Are Present in the Amino-Terminal Domain of the C-Tailed Protein SciP

ABSTRACT During their synthesis, the C-tailed membrane proteins expose the membrane-spanning segment late from the ribosome and consequently can insert into the membrane only posttranslationally. However, the C-tailed type 6 secretion system (T6SS) component SciP uses the bacterial signal recognition particle (SRP) system for membrane targeting, which operates cotranslationally. Analysis of possible sequence regions in the amino-terminal part of the protein revealed two candidates that were then tested for whether they function as SRP signal peptides. Both sequences were tested positive as synthetic peptides for binding to SRP. In addition, purified ribosomes with stalled nascent chains exposing either sequence were capable of binding to SRP and SRP-FtsY complexes with high affinity. Together, the data suggest that both peptides can serve as an SRP signal sequence promoting an early membrane targeting of SciP during its synthesis. Like observed for multispanning membrane proteins, the two cytoplasmic SRP signal sequences of SciP may also facilitate a retargeting event, making the targeting more efficient. IMPORTANCE C-tail proteins are anchored in the inner membrane with a transmembrane segment at the C terminus in an N-in/C-out topology. Due to this topology, membrane insertion occurs only posttranslationally. Nevertheless, the C-tail-anchored protein SciP is targeted cotranslationally by SRP. We report here that two amino-terminal hydrophobic stretches in SciP are individually recognized by SRP and target the nascent protein to FtsY. The presence of two signal sequences may enable a retargeting mechanism, as already observed for multispanning membrane proteins, to make the posttranslational insertion of SciP by YidC more efficient.