Dissecting the role of conserved box C/D sRNA sequences in di-sRNP assembly and function

In all three kingdoms of life, nucleotides in ribosomal RNA (rRNA) are post-transcriptionally modified. One type of chemical modification is 2′-O-ribose methylation, which is, in eukaryotes and archaea, performed by box C/D small ribonucleoproteins (box C/D sRNPs in archaea) and box C/D small nucleolar ribonucleoproteins (box C/D snoRNPs in eukaryotes), respectively. Recently, the first structure of any catalytically active box C/D s(no)RNP determined by electron microscopy and single particle analysis surprisingly demonstrated that they are dimeric RNPs. Mutational analyses of the Nop5 protein interface suggested that di-sRNP formation is also required for the in vitro catalytic activity. We have now analyzed the functional relevance of the second interface, the sRNA interface, within the box C/D di-sRNP. Mutations in conserved sequence elements of the sRNA, which allow sRNP assembly but which severely interfere with the catalytic activity of box C/D sRNPs, prevent formation of the di-sRNP. In addition, we can observe the dimeric box C/D sRNP architecture with a different box C/D sRNP, suggesting that this architecture is conserved. Together, these results provide further support for the functional relevance of the di-sRNP architecture and also provide a structural explanation for the observed defects in catalysis of 2′-O-ribose methylation

Rethinking origin licensing

Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication

Port Security: The Terrorist Naval Mine/Underwater Improvised Explosive Device Threat

Terrorist naval mines/underwater improvised explosive devices (M/UWIEDs) are a threat to U.S. maritime ports, and could cause economic damage, panic, and mass casualties. The purpose of this case study was to examine this threat and propose reforms that improve port security management. The study aligned with the mission area analysis objective of identifying and assessing potential terrorist threats in order to preempt and prevent attacks. Von Bertalanffy\u27s general systems theory was the framework for research questions, which focused on improvements in port security management to mitigate the threat of terrorist M/UWIEDs. Data collection included a document content analysis of open source/nonclassified crime reports, government threat assessments, and legislation; physical artifacts (port infrastructure) information; policy papers; maps, satellite imagery, and navigational charts; peer-reviewed academic literature; and direct observation of 2 California-based maritime ports and an inspection of their physical artifacts. Data were organized by general themes; coded axially and selectively; and analyzed by phrases, topics, and words associated with minelaying, mine countermeasures, and port security. Key findings were that, since 9/11, overall port security has improved, although there has been little progress in countering the threat presented by M/UWIEDs. Further, vulnerabilities exist that terrorists who seek to commit an M/UWIED attack or campaign could misuse. The findings from this study contribute to positive social change by providing data to key stakeholders responsible for counterterrorism, mine warfare, and port security, thereby contributing to overall U.S. homeland security

Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase

RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP and the ATPase cycle fuel the work performed by helicases is not completely clear. The hepatitis C virus RNA helicase, NS3, is an important model system for this class of enzymes. NS3 binding to a single-/double-strand RNA or DNA junction leads to ATP-independent melting of the duplex and formation of a complex capable of ATP-dependent unwinding by using a spring-loaded mechanism. We have established an RNA substrate for NS3 that can be unwound in a single sub-step. Our studies are consistent with a model in which a single ATP binding and/or hydrolysis event sets the unwinding spring and phosphate dissociation contributes to release of the spring, thereby driving the power stroke used for unwinding

Test results from a uranium hadron calorimeter using wire chamber readout

A uranium gas sampling calorimeter has been tested with electrons and pions between 1 and 50 GeV. A comparative evaluation of the response and the resolution for proportional and streamer mode operation of the gas wire chamber detectors is given for two different gas mixtures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26795/1/0000351.pd

An Endoribonuclease Functionally Linked to Perinuclear mRNP Quality Control Associates with the Nuclear Pore Complexes

Nuclear mRNA export is a crucial step in eukaryotic gene expression, which is in yeast coupled to cotranscriptional messenger ribonucleoprotein particle (mRNP) assembly and surveillance. Several surveillance systems that monitor nuclear mRNP biogenesis and export have been described, but the mechanism by which the improper mRNPs are recognized and eliminated remains poorly understood. Here we report that the conserved PIN domain protein Swt1 is an RNA endonuclease that participates in quality control of nuclear mRNPs and can associate with the nuclear pore complex (NPC). Swt1 showed endoribonuclease activity in vitro that was inhibited by a point mutation in the predicted catalytic site. Swt1 lacked clear sequence specificity but showed a strong preference for single-stranded regions. Genetic interactions were found between Swt1 and the THO/TREX and TREX-2 complexes, and with components of the perinuclear mRNP surveillance system, Mlp1, Nup60, and Esc1. Inhibition of the nuclease activity of Swt1 increased the levels and cytoplasmic leakage of unspliced aberrant pre-mRNA, and induced robust nuclear poly(A)+ RNA accumulation in mlp1Δ and esc1Δ strains. Overexpression of Swt1 also caused strong nuclear poly(A)+ RNA accumulation. Swt1 is normally distributed throughout the nucleus and cytoplasm but becomes concentrated at nuclear pore complexes (NPCs) in the nup133Δ mutant, which causes NPC clustering and defects in mRNP export. The data suggest that Swt1 endoribonuclease might be transiently recruited to NPCs to initiate the degradation of defective pre-mRNPs or mRNPs trapped at nuclear periphery in order to avoid their cytoplasmic export and translation

Genetic platelet depletion is superior in platelet transfusion compared to current models

Genetically modified mice have advanced our knowledge on platelets in hemostasis and beyond tremendously. However, mouse models harbor certain limitations, including availability of platelet specific transgenic strains, and off-target effects on other cell types. Transfusion of genetically modified platelets into thrombocytopenic mice circumvents these problems. Additionally, ex vivo treatment of platelets prior to transfusion eliminates putative side effects on other cell types. Thrombocytopenia is commonly induced by administration of anti-platelet antibodies, which opsonize platelets to cause rapid clearance. However, antibodies do not differentiate between endogenous or exogenous platelets, impeding transfusion efficacy. In contrast, genetic depletion with the inducible diphtheria toxin receptor (iDTR) system induces thrombocytopenia via megakaryocyte ablation without direct effects on circulating platelets. We compared the iDTR system with antibody-based depletion methods regarding their utility in platelet transfusion experiments, outlining advantages and disadvantages of both approaches. Antibodies led to thrombocytopenia within two hours and allowed the dose-dependent adjustment of the platelet count. The iDTR model caused complete thrombocytopenia within four days, which could be sustained for up to 11 days. Neither platelet depletion approach caused platelet activation. Only the iDTR model allowed efficient platelet transfusion by keeping endogenous platelet levels low and maintaining exogenous platelet levels over longer time periods, thus providing clear advantages over antibody-based methods. Transfused platelets were fully functional in vivo, and our model allowed examination of transgenic platelets. Using donor platelets from already available genetically modified mice or ex vivo treated platelets, may decrease the necessity of platelet-specific mouse strains, diminishing off-target effects and thereby reducing animal numbers

The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing

The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5′-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit