Structure-Templated Predictions of Novel Protein Interactions from Sequence Information

The multitude of functions performed in the cell are largely controlled by a set of carefully orchestrated protein interactions often facilitated by specific binding of conserved domains in the interacting proteins. Interacting domains commonly exhibit distinct binding specificity to short and conserved recognition peptides called binding profiles. Although many conserved domains are known in nature, only a few have well-characterized binding profiles. Here, we describe a novel predictive method known as domain–motif interactions from structural topology (D-MIST) for elucidating the binding profiles of interacting domains. A set of domains and their corresponding binding profiles were derived from extant protein structures and protein interaction data and then used to predict novel protein interactions in yeast. A number of the predicted interactions were verified experimentally, including new interactions of the mitotic exit network, RNA polymerases, nucleotide metabolism enzymes, and the chaperone complex. These results demonstrate that new protein interactions can be predicted exclusively from sequence information

MicroRNAs from saliva of anopheline mosquitoes mimic human endogenous miRNAs and may contribute to vector-host-pathogen interactions

During blood feeding haematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract host haemostasis, inflammation and immunity. However, animal body fluids are known to also carry miRNAs. To get insights into saliva and salivary gland miRNA repertoires of the African malaria vector Anopheles coluzzii we used small RNA-Seq and identified 214 miRNAs, including tissue-enriched, sex-biased and putative novel anopheline miRNAs. Noteworthy, miRNAs were asymmetrically distributed between saliva and salivary glands, suggesting that selected miRNAs may be preferentially directed toward mosquito saliva. The evolutionary conservation of a subset of saliva miRNAs in Anopheles and Aedes mosquitoes, and in the tick Ixodes ricinus, supports the idea of a non-random occurrence pointing to their possible physiological role in blood feeding by arthropods. Strikingly, eleven of the most abundant An. coluzzi saliva miRNAs mimicked human miRNAs. Prediction analysis and search for experimentally validated targets indicated that miRNAs from An. coluzzii saliva may act on host mRNAs involved in immune and inflammatory responses. Overall, this study raises the intriguing hypothesis that miRNAs injected into vertebrates with vector saliva may contribute to host manipulation with possible implication for vector-host interaction and pathogen transmission

Structure and Function of a Mycobacterial NHEJ DNA Repair Polymerase

Non homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks in prokaryotes requires Ku and a specific multidomain DNA ligase (LigD). We present crystal structures of the primase/polymerisation domain (PolDom) of Mycobacterium tuberculosis LigD, alone and complexed with nucleotides. The PolDom structure combines the general fold of the archaeo-eukaryotic primase (AEP) superfamily with additional loops and domains that together form a deep cleft on the surface, likely used for DNA binding. Enzymatic analysis indicates that the PolDom of LigD, even in the absence of accessory domains and Ku proteins, has the potential to recognise DNA end-joining intermediates. Strikingly, one of the main signals for the specific and efficient binding of PolDom to DNA is the presence of a 5'-phosphate group, located at the single/double-stranded junction at both gapped and 3'-protruding DNA molecules. Although structurally unrelated, Pol lambda and Pol mu, the two eukaryotic DNA polymerases involved in NHEJ, are endowed with a similar capacity to bind a 5'-phosphate group. Other properties that are beneficial for NHEJ, such as the ability to generate template distortions and realignments of the primer, displayed by Pol lambda and Pol mu, are shared by the PolDom of bacterial LigD. In addition, PolDom can perform non-mutagenic translesion synthesis on termini containing modified bases. Significantly, ribonucleotide insertion appears to be a recurrent theme associated with NHEJ, maximised in this case by the deployment of a dedicated primase, although its in vivo relevance is unknown

An Alternative View of the Templates and Functions of RNA Polymerase II

R N A polymerase II (pol II) has been implicated in the RNA-templated RNA synthesis during replication of some Viroids and Hepatitis Delta Virus (HDV). In this study HDV RNA-templated pol II transcription was examined in vitro, using HeLa cell nuclear extracts (NE). Under standard conditions for the DNAtemplated pol II transcription, a segment of the antigenomic (AG) HDV RNA encompassing the left-hand tip region of the HDV rod-like structure, serves as a template for efficient transcription. The transcription reaction is highly sensitive to a-amanitin in HeLa cell NE, but it is partially resistant to this toxin in NE from PMG cells that contain an a-amanitin resistant allele of the pol II largest subunit, strongly suggesting pol II involvement in the process. Direct RNase A, T1 and RNAse H digestion analyses demonstrate that the product of the HDV RNA dependent pol II transcription represents a chimeric molecule, in which the pol II transcript is covalently attached to the 5\u27 half of the AG RNA template. Such a chimeric RNA product is generated by a cleavage of the template at a specific site followed by transcription that uses the new end as a primer. Pol II transcription of the RNA template proceedes with high fidelity but appears to pause after -40 transcribed nucleotides. A viral protein, delta antigen (HDAg), stimulates pol II elongation on the RNA template. The initiation of HDV RNA transcription that involves cleavage of the RNA template may be mechanistically similar to the endonucleolytic cleavage of the nascent transcript that, under certain conditions, occurs during the elongation phase of pol II transcription on a \u27typical\u27 DNA templates. Mutational and secondary structure mapping analyses of HDV RNA templates demonstrate that the secondary structure, rather than the primary sequence, specifies the functional pol II transcription templates. The secondary structure in the left-hand terminal hairpin of the HDV RNA also modulates HDV replication in vivo, in COS7 and HeLa cells. The correlation between the effects of secondary structure alterations on the efficiency of pol II transcription in vitro and HDV replication in vivo suggests that the observed RNA-templated pol II transcription in vitro reflects an important regulatory step of the HDV replication cycle. Based on these findings, a modified pol ll-mediated rolling-circle model for HDV replication is proposed. It involves initiation of transcription by cleavage of one RNA template, followed by pol II switching to a new circular template. The model also suggests that, both the initiation and the template switching steps are regulated by specific secondary structure elements in the terminal hairpin of HDV RNA

tRNA signatures reveal polyphyletic origins of streamlined SAR11 genomes among the alphaproteobacteria

Phylogenomic analyses are subject to bias from compositional convergence and noise from horizontal gene transfer (HGT). Compositional convergence is a likely cause of controversy regarding phylogeny of the SAR11 group of Alphaproteobacteria that have extremely streamlined, A+T-biased genomes. While careful modeling can reduce artifacts caused by convergence, the most consistent and robust phylogenetic signal in genomes may lie distributed among encoded functional features that govern macromolecular interactions. Here we develop a novel phyloclassification method based on signatures derived from bioinformatically defined tRNA Class-Informative Features (CIFs). tRNA CIFs are enriched for features that underlie tRNA-protein interactions. Using a simple tRNA-CIF-based phyloclassifier, we obtained results consistent with those of bias-corrected whole proteome phylogenomic studies, rejecting monophyly of SAR11 and affiliating most strains with Rhizobiales with strong statistical support. Yet SAR11 and Rickettsiales tRNA genes share distinct patterns of A+T-richness, as expected from their elevated genomic A+T compositions. Using conventional supermatrix methods on total tRNA sequence data, we could recover the artifactual result of a monophyletic SAR11 grouping with Rickettsiales. Thus tRNA CIF-based phyloclassification is more robust to base content convergence than supermatrix phylogenomics on whole tRNA sequences. Also, given the notoriously promiscuous HGT of aminoacyl-tRNA synthetases, tRNA CIF-based phyloclassification may be relatively robust to HGT of network components. We describe how unique features of tRNA-protein interaction networks facilitate the mining of traits governing macromolecular interactions from genomic data, and discuss why interaction-governing traits may be especially useful to solve difficult problems in microbial classification and phylogeny