Aberrant B cell repertoire selection associated with HIV neutralizing antibody breadth.

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.

An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.

Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Protection from feed-forward amplification in an amplified RNAi mechanism.

The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA-directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in Caenorhabditis elegans, we find that feed-forward amplification is limited by biosynthetic and structural distinctions at the RNA level between (1) triggers that can produce amplification and (2) siRNA products of the amplification reaction. By assuring that initial (primary) siRNAs can act as triggers but not templates for activation, and that the resulting (secondary) siRNAs can enforce gene silencing on additional targets without unbridled trigger amplification, the system achieves substantial but fundamentally limited signal amplification.

A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

DNA Polymerase Diversity Reveals Multiple Incursions of Polintons During Nematode Evolution.

Polintons are double-stranded DNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family but encode a distinct protein-primed DNA polymerase B (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting interphylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of an HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.

PLP-1 is essential for germ cell development and germline gene silencing in Caenorhabditiselegans.

The germline genome is guarded against invading foreign genetic elements by small RNA-dependent gene-silencing pathways. Components of these pathways localize to, or form distinct aggregates in the vicinity of, germ granules. These components and their dynamics in and out of granules are currently being intensively studied. Here, we report the identification of PLP-1, a Caenorhabditiselegans protein related to the human single-stranded nucleic acid-binding protein Pur-alpha, as a component of germ granules in C. elegans We show that PLP-1 is essential for silencing different types of transgenes in the germ line and for suppressing the expression of several endogenous genes controlled by the germline gene-silencing pathways. Our results reveal that PLP-1 functions downstream of small RNA biogenesis during initiation of gene silencing. Based on these results and the earlier findings that Pur-alpha proteins interact with both RNA and protein, we propose that PLP-1 couples certain RNAs with their protein partners in the silencing complex. PLP-1 orthologs localized on RNA granules may similarly contribute to germline gene silencing in other organisms.

Restriction Endonuclease-Based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing.

Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus enriching for undigested DNA from the organism of interest. As a proof of concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. IMPORTANCE Pathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA, which may escape unnoticed in metagenomic samples.

Context-dependent DNA polymerization effects can masquerade as DNA modification signals.

BACKGROUND: Single molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach uses a Single Molecule Real Time (SMRT)-sequencing measurement of time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD). RESULTS: In this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Adenine modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples. CONCLUSION: The latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome.

Recompleting the Caenorhabditis elegans genome.

Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted ≥53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology.

Translation readthrough mitigation.

A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR­Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3' untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3' UTR sequences to reduce mature protein expression in tissue culture assays, including 3' UTR sequences from the hypomorphic 'Constant Spring' haemoglobin stop codon variant. We suggest that 3' UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

Ribosome clearance during RNA interference.

In the course of identifying and cleaving RNA, the RNAi machinery must encounter and contend with the megadalton-sized ribosomes that carry out translation. We investigated this interface by examining the fate of actively translated mRNAs subjected to RNAi in C. elegans Quantifying RNA levels (RNA-seq) and ongoing translation (Ribo-seq), we found there is a greater fold repression of ongoing translation than expected from loss of RNA alone, observing stronger translation repression relative to RNA repression for multiple, independent double-stranded RNA triggers, and for multiple genes. In animals that lack the RNA helicase SKI complex and the ribosome rescue factor PELOTA, ribosomes stall on the 3' edges of mRNAs at and upstream of the RNAi trigger. One model to explain these observations is that ribosomes are actively cleared from mRNAs by SKI and PELO during or following mRNA cleavage. Our results expand prior studies that show a role for the SKI RNA helicase complex in removing RNA targets following RNAi in flies and plants, illuminating the widespread role of the nonstop translation surveillance in RNA silencing during RNAi. Our results are also consistent with proposals that RNAi can attack messages during active translation.

Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications.

Next-generation DNA-sequencing (NGS) technologies, which are designed to streamline the acquisition of massive amounts of sequencing data, are nonetheless dependent on various preparative steps to generate DNA fragments of required concentration, purity and average size (molecular weight). Current automated electrophoresis systems for DNA- and RNA-sample quality control, such as Agilent's Bioanalyzer® and TapeStation® products, are costly to acquire and use; they also provide limited information for samples having broad size distributions. Here, we describe a software tool that helps determine the size distribution of DNA fragments in an NGS library, or other DNA sample, based on gel-electrophoretic line profiles. The software, developed as an ImageJ plug-in, allows for straightforward processing of gel images, including lane selection and fitting of univariate functions to intensity distributions. The user selects the option of fitting either discrete profiles in cases where discrete gel bands are visible or continuous profiles, having multiple bands buried under a single broad peak. The method requires only modest imaging capabilities and is a cost-effective, rigorous alternative characterization method to augment existing techniques for library quality control.

Intron and gene size expansion during nervous system evolution.

BACKGROUND: The evolutionary radiation of animals was accompanied by extensive expansion of gene and genome sizes, increased isoform diversity, and complexity of regulation. RESULTS: Here we show that the longest genes are enriched for expression in neuronal tissues of diverse vertebrates and of invertebrates. Additionally, we show that neuronal gene size expansion occurred predominantly through net gains in intron size, with a positional bias toward the 5' end of each gene. CONCLUSIONS: We find that intron and gene size expansion is a feature of many genes whose expression is enriched in nervous systems. We speculate that unique attributes of neurons may subject neuronal genes to evolutionary forces favoring net size expansion. This process could be associated with tissue-specific constraints on gene function and/or the evolution of increasingly complex gene regulation in nervous systems.

Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

'Inc-miRs': functional intron-interrupted miRNA genes.

The discovery of microRNAs (miRNAs) lin-4 and let-7 as temporal regulators in Caenorhabditis elegans led to broader searches for novel miRNAs and their biological roles. Unlike protein-coding genes and some long noncoding RNAs, canonical metazoan miRNAs are not known to contain introns within their genomic precursor sequences. Because the short length of miRNAs complicates a statistically definitive assignment of split genes in RNA sequencing data sets, we took an experimental approach toward testing the compatibility of splicing and functional miRNA biogenesis. To definitively evaluate the possibility that miRNAs could derive from interrupted genes, we constructed intron-interrupted variants of C. elegans lin-4 and assayed for their miRNA-encoding capability and biological activity in the developing organism. Our studies indicate that (1) intron-containing miRNAs (inc-miRs) can be efficiently spliced and processed to produce miRNAs with normal termini, and (2) these miRNAs can be functional in full rescue of developmental phenotypes in null mutants lacking endogenous lin-4. This study provides the first evidence to support the ability of intron-interrupted miRNA precursors to produce functional regulators and identifies an additional modality available for metazoan miRNA production.

A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality.

Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals.

Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.

Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma.

Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26 nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the Argonaute ERGO-1, DICER, and a series of associated ("ERI") factors. This primary process leads to production of a much more abundant class of 22 nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA.

Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo.

Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.