Engineering of temperature- and light-switchable Cas9 variants
Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonucl...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Richter, F., Fonfara, I., Bouazza, B., Schumacher, C. H., Bratovic, M., Charpentier, E., Möglich, A. Tags: Synthetic Biology and Assembly Cloning Synthetic Biology and Bioengineering Source Type: research
Protein-DNA interfaces: a molecular dynamics analysis of time-dependent recognition processes for three transcription factors
We have studied the dynamics of three transcription factor–DNA complexes using all-atom, microsecond-scale MD simulations. In each case, the salt bridges and hydrogen bond interactions formed at the protein–DNA interface are found to be dynamic, with lifetimes typically in the range of tens to hundreds of picoseconds, although some interactions, notably those involving specific binding to DNA bases, can be a hundred times longer lived. Depending on the complex studied, this dynamics may or may not lead to the existence of distinct conformational substates. Using a sequence threading technique, it has been possi...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Etheve, L., Martin, J., Lavery, R. Tags: Structural Biology Source Type: research
hLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA
This study confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function. (Source: Nucleic Acids Research)
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Eichhorn, C. D., Chug, R., Feigon, J. Tags: Structural Biology Source Type: research
Molecular basis of cobalamin-dependent RNA modification
Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis. Our structure of QueG bound to a tRNATyr anticodon stem loop ...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Dowling, D. P., Miles, Z. D., Köhrer, C., Maiocco, S. J., Elliott, S. J., Bandarian, V., Drennan, C. L. Tags: Structural Biology Source Type: research
Structural dynamics control the MicroRNA maturation pathway
MicroRNAs (miRNAs) are crucial gene expression regulators and first-order suspects in the development and progression of many diseases. Comparative analysis of cancer cell expression data highlights many deregulated miRNAs. Low expression of miR-125a was related to poor breast cancer prognosis. Interestingly, a single nucleotide polymorphism (SNP) in miR-125a was located within a minor allele expressed by breast cancer patients. The SNP is not predicted to affect the ground state structure of the primary transcript or precursor, but neither the precursor nor mature product is detected by RT-qPCR. How this SNP modulates the...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Dallaire, P., Tan, H., Szulwach, K., Ma, C., Jin, P., Major, F. Tags: RNA characterisation and manipulation, Computational Methods, Targeted inhibition of gene function Structural Biology Source Type: research
S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells
MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP inter...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Warner, M. J., Bridge, K. S., Hewitson, J. P., Hodgkinson, M. R., Heyam, A., Massa, B. C., Haslam, J. C., Chatzifrangkeskou, M., Evans, G. J. O., Plevin, M. J., Sharp, T. V., Lagos, D. Tags: RNA Source Type: research
Cellular and molecular phenotypes depending upon the RNA repair system RtcAB of Escherichia coli
RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to su...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Engl, C., Schaefer, J., Kotta-Loizou, I., Buck, M. Tags: RNA Source Type: research
Binding properties of YjeQ (RsgA), RbfA, RimM and Era to assembly intermediates of the 30S subunit
Our understanding regarding the function of YjeQ (also called RsgA), RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in null strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that these immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particl...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Thurlow, B., Davis, J. H., Leong, V., F. Moraes, T., Williamson, J. R., Ortega, J. Tags: RNA Source Type: research
PCBP2 enables the cadicivirus IRES to exploit the function of a conserved GRNA tetraloop to enhance ribosomal initiation complex formation
The cadicivirus IRES diverges structurally from canonical Type 1 IRESs (e.g. poliovirus) but nevertheless also contains an essential GNRA tetraloop in a subdomain (d10c) that is homologous to poliovirus dIVc. In addition to canonical initiation factors, the canonical Type 1 and divergent cadicivirus IRESs require the same IRES trans-acting factor, poly(C)-binding protein 2 (PCBP2). PCBP2 has three KH domains and binds poliovirus IRES domain dIV in the vicinity of the tetraloop. How PCBP2 binds the cadicivirus IRES, and the roles of PCBP2 and the tetraloop in Type 1 IRES function are unknown. Here, directed hydroxyl radical...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Asnani, M., Pestova, T. V., Hellen, C. U. T. Tags: RNA Source Type: research
The functional requirement of two structural domains within telomerase RNA emerged early in eukaryotes
Telomerase emerged during evolution as a prominent solution to the eukaryotic linear chromosome end-replication problem. Telomerase minimally comprises the catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TR) that provides the template for telomeric DNA synthesis. While the TERT protein is well-conserved across taxa, TR is highly divergent amongst distinct groups of species. Herein, we have identified the essential functional domains of TR from the basal eukaryotic species Trypanosoma brucei, revealing the ancestry of TR comprising two distinct structural core domains that can assemble in trans with TE...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Podlevsky, J. D., Li, Y., Chen, J. J.- L. Tags: RNA Source Type: research
5-methylcytosine-sensitive variants of Thermococcus kodakaraensis DNA polymerase
DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2'-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson–Crick recognition. Here, we des...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Huber, C., von Watzdorf, J., Marx, A. Tags: Nucleic Acid Enzymes Source Type: research
Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method
In this study, we endeavored to reveal detailed structure–activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5' binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Wang, Y., Beal, P. A. Tags: Nucleic Acid Enzymes Source Type: research
VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation
The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin–Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs f...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Winther, K., Tree, J. J., Tollervey, D., Gerdes, K. Tags: Nucleic Acid Enzymes Source Type: research
Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA
Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5' end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Izumikawa, K., Yoshikawa, H., Ishikawa, H., Nobe, Y., Yamauchi, Y., Philipsen, S., Simpson, R. J., Isobe, T., Takahashi, N. Tags: Molecular Biology Source Type: research
A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8
Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential f...
Source: Nucleic Acids Research - November 28, 2016 Category: Research Authors: Sztuba-Solinska, J., Diaz, L., Kumar, M. R., Kolb, G., Wiley, M. R., Jozwick, L., Kuhn, J. H., Palacios, G., Radoshitzky, S. R., J. Le Grice, S. F., Johnson, R. F. Tags: Molecular Biology Source Type: research
