Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA.

Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.

Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation.

BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults. RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages. CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.

The structural basis for the difference in absorbance spectra for the FMO antenna protein from various green sulfur bacteria.

The absorbance spectrum of the Fenna-Matthews-Olson protein--a component of the antenna system of Green Sulfur Bacteria--is always one of two types, depending on the species of the source organism. The FMO from Prosthecochloris aestuarii 2K has a spectrum of type 1 while that from Chlorobaculum tepidum is of type 2. The previously reported crystal structures for these two proteins did not disclose any rationale that would explain their spectral differences. We have collected a 1.3 A X-ray diffraction dataset of the FMO from Prosthecochloris aestuarii 2K, which has allowed us to identify an additional Bacteriochlorophyll-a molecule with chemical attachments to both sides of the central magnesium atom. A new analysis of the previously published X-ray data for the Chlorobaculum tepidum FMO shows the presence of a Bacteriochlorophyll-a molecule in an equivalent location but with a chemical attachment from only one side. This difference in binding is shown to be predictive of the spectral type of the FMO.

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N.

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.

Applying thiouracil tagging to mouse transcriptome analysis.

Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type-specific RNA. TU tagging enables the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, as well as the identification of actively transcribed RNAs and not preexisting transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on the purification of tagged ribosomes or nuclei, TU tagging provides a direct examination of transcriptional regulation. We describe how to (i) deliver 4-thiouracil to transgenic mice to thio-label cell lineage-specific transcripts, (ii) purify TU-tagged RNA and prepare libraries for Illumina sequencing and (iii) follow a straightforward bioinformatics workflow to identify cell type-enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in 1 d, RNA-seq libraries can be generated within 2 d and, after sequencing, an initial bioinformatics analysis can be completed in 1 additional day.

Structure of the nucleocapsid-binding domain from the mumps virus polymerase; an example of protein folding induced by crystallization.

The human pathogen mumps virus, like all paramyxoviruses, encodes a polymerase responsible for virally directed RNA synthesis. The template for the polymerase is the nucleocapsid, a filamentous protein-RNA complex harboring the viral genome. Interaction of the polymerase and the nucleocapsid is mediated by a small domain tethered to the end of the phosphoprotein (P), one of the polymerase subunits. We report the X-ray crystal structure of this region of mumps virus P (the nucleocapsid-binding domain, or NBD, amino acids 343-391). The mumps P NBD forms a compact bundle of three alpha-helices within the crystal, a fold apparently conserved across the Paramyxovirinae. In solution, however, the domain exists in the molten globule state. This is demonstrated through application of differential scanning calorimetry, circular dichroism spectroscopy, NMR spectroscopy, and dynamic light scattering. While the mumps P NBD is compact and has persistent secondary structure, it lacks a well-defined tertiary structure under normal solution conditions. It can, however, be induced to fold by addition of a stabilizing methylamine cosolute. The domain provides a rare example of a molten globule that can be crystallized. The structure that is stabilized in the crystal represents the fully folded state of the domain, which must be transiently realized during binding to the viral nucleocapsid. While the intermolecular forces that govern the polymerase-nucleocapsid interaction appear to be different in measles, mumps, and Sendai viruses, for each of these viruses, polymerase translocation involves the coupled binding and folding of protein domains. In all cases, we suggest that this will result in a weak-affinity protein complex with a short lifetime, which allows the polymerase to take rapid steps forward.

Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site.

Aminopeptidase N from Escherichia coli is a major metalloprotease that participates in the controlled hydrolysis of peptides in the proteolytic pathway. Determination of the 870-aa structure reveals that it has four domains similar to the tricorn-interacting factor F3. The thermolysin-like active site is enclosed within a large cavity with a volume of 2,200 A(3), which is inaccessible to substrates except for a small opening of approximately 8-10 A. The substrate-based inhibitor bestatin binds to the protein with minimal changes, suggesting that this is the active form of the enzyme. The previously described structure of F3 had three distinct conformations that were described as "closed," "intermediate," and "open." The structure of aminopeptidase N from E. coli, however, is substantially more closed than any of these. Taken together, the results suggest that these proteases, which are involved in intracellular peptide degradation, prevent inadvertent hydrolysis of inappropriate substrates by enclosing the active site within a large cavity. There is also some evidence that the open form of the enzyme, which admits substrates, remains inactive until it adopts the closed form.

Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins.

We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C. Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

Long-distance conformational changes in a protein engineered by modulated sequence duplication.

There are few, if any, known instances in which a biological signal is transmitted via a large conformational change through the body of a protein. We describe here a mutant of T4 lysozyme that was engineered to permit structural change at a distance. The design uses a tandem sequence repeat that makes it possible to transmit large-scale structural changes from one end of an alpha-helix to the other over a distance of 17-25 A. The method should be of general applicability and may make it possible to introduce a mutation at one site in a protein that will induce large-scale changes in the structure at a spatially remote site.

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding.

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.

Structural basis for the attachment of a paramyxoviral polymerase to its template.

The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding domain. We have investigated the interaction between the binding domain from measles P (amino acids 457-507) and the sequence it recognizes within measles N (amino acids 477-505). By using both solution NMR spectroscopy and x-ray crystallography, we show that N(487-503) binds as a helix to the surface created by the second (alpha2) and third (alpha3) helices of P(457-507), in an orientation parallel to the helix alpha3, creating a four-helix bundle. The binding interface is tightly packed and dominated by hydrophobic amino acids. Binding and folding of N(487-503) are coupled. However, when not bound to P, N(487-503) does not resemble a statistical random coil but instead exists in a loosely structured state that mimics the bound conformation. We propose that before diffusional encounter, the ensemble of accessible conformations for N(487-503) is biased toward structures capable of binding P, facilitating rapid association of the two proteins. This study provides a structural analysis of polymerase-template interactions in a paramyxovirus and presents an example of a protein-protein interaction that must be only transiently maintained as part of its normal function.