Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome.

Anelloviruses are a ubiquitous component of healthy human viromes and remain highly prevalent after being acquired early in life. The full extent of "anellome" diversity and its evolutionary dynamics remain unexplored. We employed in-depth sequencing of blood-transfusion donor(s)-recipient pairs coupled with public genomic resources for a large-scale assembly of anellovirus genomes and used the data to characterize global and personal anellovirus diversity through time. The breadth of the anellome is much greater than previously appreciated, and individuals harbor unique anellomes and transmit lineages that can persist for several months within a diverse milieu of endemic host lineages. Anellovirus sequence diversity is shaped by extensive recombination at all levels of divergence, hindering traditional phylogenetic analyses. Our findings illuminate the transmission dynamics and vast diversity of anelloviruses and set the foundation for future studies to characterize their biology.

Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations.

Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.

What would you do if you could sequence everything?

It could be argued that the greatest transformative aspect of the Human Genome Project has been not the sequencing of the genome itself, but the resultant development of new technologies. A host of new approaches has fundamentally changed the way we approach problems in basic and translational research. Now, a new generation of high-throughput sequencing technologies promises to again transform the scientific enterprise, potentially supplanting array-based technologies and opening up many new possibilities. By allowing DNA/RNA to be assayed more rapidly than previously possible, these next-generation platforms promise a deeper understanding of genome regulation and biology. Significantly enhancing sequencing throughput will allow us to follow the evolution of viral and bacterial resistance in real time, to uncover the huge diversity of novel genes that are currently inaccessible, to understand nucleic acid therapeutics, to better integrate biological information for a complete picture of health and disease at a personalized level and to move to advances that we cannot yet imagine.

A mechanism of translational repression by competition of Paip2 with eIF4G for poly(A) binding protein (PABP) binding.

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by the cap-binding protein eIF4E, the poly(A) binding protein (PABP), and eIF4G, a scaffolding protein that bridges between eIF4E and PABP to bring about the circularization of the mRNA. The translational repressor, Paip2 (PABP-interacting protein 2), inhibits translation by promoting the dissociation of PABP from poly(A). Here we report on the existence of an alternative mechanism by which Paip2 inhibits translation by competing with eIF4G for binding to PABP. We demonstrate that Paip2 can abrogate the translational activity of PABP, which is tethered to the 3' end of the mRNA. Thus, Paip2 can inhibit translation by a previously unrecognized mechanism, which is independent of its ability to disrupt PABP-poly(A) interaction.

Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2.

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3' poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5' end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.

Poly(A)-binding protein binds to A-rich sequences via RNA-binding domains 1+2 and 3+4.

Poly(A) binding protein (PABP) binds non-protein-coding BC1 RNA and BC200 RNA, which contain adenosine-rich domains. Two combinations of the four PABP RNA recognition motifs (RRMs), RRMs 1+2 and RRMs 3+4, bind with very strong affinities to various transcripts with long stretches of adenosine residues, whereas RRMs 2+3 bind weakly. While RRMs 1+2 preferentially bind to stretches that contain only adenosines, RRMs 3+4 exhibit relatively high affinities towards sequences that are interspersed with other nucleotides. Binding studies with oligoribonucleotide(A)(65) and oligoribonucleotide(A)(25) showed that the shorter RNA is not an ideal substrate for binding studies to model the interactions with mRNAs, which in general harbor long poly(A) tails.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant.

Messenger RNA decay mediated by the c-fos major protein coding-region determinant of instability (mCRD) is a useful system for studying translationally coupled mRNA turnover. Among the five mCRD-associated proteins identified previously, UNR was found to be an mCRD-binding protein and also a PABP-interacting protein. Interaction between UNR and PABP is necessary for the full destabilization function of the mCRD. By testing different classes of mammalian poly(A) nucleases, we identified CCR4 as a poly(A) nuclease involved in the mCRD-mediated rapid deadenylation in vivo and also associated with UNR. Blocking either translation initiation or elongation greatly impeded poly(A) shortening and mRNA decay mediated by the mCRD, demonstrating that the deadenylation step is coupled to ongoing translation of the message. These findings suggest a model in which the mCRD/UNR complex serves as a "landing/assembly" platform for formation of a deadenylation/decay mRNA-protein complex on an mCRD-containing transcript. The complex is dormant prior to translation. Accelerated deadenylation and decay of the transcript follows ribosome transit through the mCRD. This study provides new insights into a mechanism by which interplay between mRNA turnover and translation determines the lifespan of an mCRD-containing mRNA in the cytoplasm.

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase.

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.

Paip1 interacts with poly(A) binding protein through two independent binding motifs.

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.