Nuclear Surveillance Research Articles

RNAi-Dependent and -Independent RNA Turnover Mechanisms Contribute to Heterochromatic Gene Silencing

In fission yeast, the RNAi pathway is required for heterochromatin-dependent silencing of transgene insertions at centromeric repeats and acts together with other pathways to silence transgenes at the silent mating-type locus. Here, we show that transgene transcripts at centromeric repeats are processed into siRNAs and are therefore direct targets of RNAi. Furthermore, we show that Cid14, a member of the Trf4/5 family of poly(A) polymerases, has poly(A) polymerase activity that is required for heterochromatic gene silencing. Surprisingly, while siRNA levels in cid14Delta cells are dramatically reduced, the structural integrity of heterochromatin appears to be preserved. Cid14 resides in a complex similar to the TRAMP complex found in budding yeast, which is part of a nuclear surveillance mechanism that degrades aberrant transcripts. Our findings indicate that polyadenylation by a TRAMP-like complex contributes to robust silencing of heterochromatic genes in fission yeast via the recruitment of the exosome and/or the RNAi machinery.

Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' --> 5' exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.

Differential Recruitment of the Splicing Machinery during Transcription Predicts Genome-Wide Patterns of mRNA Splicing

The splicing machinery associates with genes to facilitate efficient cotranscriptional mRNA processing. We have mapped these associations by genome localization analysis to ascertain how splicing is achieved and regulated on a system-wide scale. Our data show that factors important for intron recognition sample nascent mRNAs and are retained specifically at intron-containing genes via RNA-dependent interactions. Spliceosome assembly proceeds cotranscriptionally but completes posttranscriptionally in most cases. Some intron-containing genes were not bound by the spliceosome, including several developmentally regulated genes. On this basis, we predicted and verified regulated splicing and observed a role for nuclear mRNA surveillance in monitoring those events. Finally, we present evidence that cotranscriptional processing events determine the recruitment of specific mRNA export factors. Broadly, our results provide mechanistic insights into the coordinated regulation of transcription, mRNA processing, and nuclear export in executing complex gene expression programs.

Identification of a Rapid Mammalian Deadenylation-Dependent Decay Pathway and Its Inhibition by a Viral RNA Element

Cellular RNAs are subject to quality-control pathways that insure the fidelity of gene expression. We previously identified a 79 nt element, the ENE, that is essential for the nuclear accumulation of a viral polyadenylated nuclear (PAN) RNA. Here, we show that intron-less polyadenylated transcripts such as PAN RNA and beta-globin cRNA exhibit two-component exponential decay kinetics in which some transcripts are rapidly degraded (t(1/2) = approximately 15 min) while others decay more slowly (t(1/2) = approximately 3 hr). Inclusion of the ENE protects such transcripts from rapid decay in a poly(A)-dependent fashion. The ENE inhibits deadenylation and decay in nuclear extract and prevents deadenylation of naked RNA by a purified deadenylase, likely through snoRNA-like intramolecular hybridization with the poly(A) tail. The ENE causes increased accumulation of splicing-defective beta-globin pre-mRNAs in vivo. We propose that the ENE-controlled rapid-decay mechanism for polyadenylated transcripts comprises a nuclear pre-mRNA surveillance system in mammalian cells.

A niching genetic algorithm applied to a nuclear power plant auxiliary feedwater system surveillance tests policy optimization

This article extends previous efforts on genetic algorithms (GAs) applied to a nuclear power plant (NPP) auxiliary feedwater system (AFWS) surveillance tests policy optimization. We introduce the application of a niching genetic algorithm (NGA) to this problem and compare its performance to previous results. The NGA maintains a populational diversity during the search process, thus promoting a greater exploration of the search space. The optimization problem consists in maximizing the system’s average availability for a given period of time, considering realistic features such as: (i) aging effects on standby components during the tests; (ii) revealing failures in the tests implies on corrective maintenance, increasing outage times; (iii) components have distinct test parameters (outage time, aging factors, etc.) and (iv) tests are not necessarily periodic. We find that the NGA performs better than the conventional GA and the island GA due to a greater exploration of the search space.

ALARA Considerations for the Whole Body Neutron Irradiation Facility Source Removal Project at Brookhaven National Laboratory

This paper describes the activities that were involved with the safe removal of fourteen PuBe sources from the Brookhaven National Laboratory (BNL) Whole Body Neutron Irradiation Facility (WBNIF). As part of a Department of Energy and BNL effort to reduce the radiological inventory, the WBNIF was identified as having no future use. In order to deactivate the facility and eliminate the need for nuclear safety management and long-term surveillance, it was decided to remove the neutron sources and dismantle the facility. In addition, the sources did not have DOT Special Form documentation so they would need to be encapsulated once removed for offsite storage or disposal. The planning and the administrative as well as engineering controls put in place enabled personnel to safely remove and encapsulate the sources while keeping exposure as low as reasonably achievable (ALARA).

Rapid tRNA Decay Can Result from Lack of Nonessential Modifications

The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.

The Challenge of Viral snRNPs

Some gammaherpesviruses encode nuclear noncoding RNAs (ncRNAs) that assemble with host proteins. Their conservation and abundance implies that they serve important functions for the virus. This paper focuses on our studies of three classes of nuclear noncoding herpesvirus RNAs. (1) EBERs 1 and 2 are expressed by Epstein-Barr virus in latent infection of human B lymphocytes. Recent studies revealed three sites on EBER1 that associate with ribosomal protein L22. In addition, heterokaryon assays have definitively shown that both EBERs are confined to the nucleus, arguing that their contribution to viral latency is purely nuclear. (2) HSURs 1-7 are U RNAs encoded by Herpesvirus saimiri, which causes aggressive T-cell leukemias and lymphomas. Comparison of monkey T cells transformed with wild-type or mutant virus lacking HSURs 1 and 2 revealed significant changes in host mRNAs implicated in T-cell signaling. (3) PAN is a 1-kb polyadenylated RNA that accumulates in the nucleus of Kaposi's sarcoma-associated herpesvirus lytically infected cells. A novel element, the ENE, is essential for its high accumulation. Recent results indicate that the ENE functions to counteract poly(A)-dependent RNA degradation, which we propose contributes to nuclear surveillance of mRNA transcripts in mammalian cells. Continuing studies of these viral RNAs will provide insights into both cellular and viral gene expression.

A nuclear surveillance pathway for mRNAs with defective polyadenylation.

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

RNA Turnover: Unexpected Consequences of Being Tailed

In eukaryotic cells, the 3' poly(A) tails found on mRNA influence their stability and translation. The discovery of a second nuclear poly(A) polymerase complex has fueled a series of reports defining a new and unexpected role for 3' end poly(A) tails in the nuclear surveillance and turnover of noncoding RNAs and intergenic mRNAs of unknown function.

Regulation and Surveillance of Normal and 3′-Extended Forms of the Yeast Aci-reductone Dioxygenase mRNA by RNase III Cleavage and Exonucleolytic Degradation

Aci-reductone dioxygenases are key enzymes in the methionine salvage pathway. The mechanisms by which the expression of this important class of enzymes is regulated are poorly understood. Here we show that the expression of the mRNA encoding the yeast aci-reductone dioxygenase ADI1 is controlled post-transcriptionally by RNase III cleavage. Cleavage occurs in a large bipartite stem loop structure present in the open reading frame region of the ADI1 mRNA. The ADI1 mRNA is up-regulated in the absence of the yeast orthologue of RNase III Rnt1p or of the 5' --> 3' exonucleases Xrn1p and Rat1p. 3'-Extended forms of this mRNA, including a polycistronic mRNA ADI1-YMR010W mRNA, also accumulate in cells lacking Rnt1p, Xrn1p, and Rat1p or the nuclear exosome component Rrp6p, suggesting that these 3'-extended forms are subject to nuclear surveillance. We show that the ADI1 mRNA is up-regulated under heat shock conditions in a Rnt1p-independent manner. We propose that Rnt1p cleavage targets degradation of the ADI1 mRNA to prevent its expression prior to heat shock conditions and that RNA surveillance by multiple ribonucleases helps prevent accumulation of aberrant 3'-extended forms of this mRNA that arise from intrinsically inefficient 3'-processing signals.

Premature termination codons do not affect the rate of splicing of neighboring introns.

Introduction of a premature termination codon (PTC) into an exon of a gene can lead to nonsense-mediated decay of the mRNA, which is best characterized as a cytoplasmic event. However, increasing evidence has suggested that PTCs may also influence the nuclear processing of an RNA transcript, leading to models of nuclear surveillance perhaps involving translating nuclear ribosomes. We used quantitative RT-PCR to measure the in vivo steady-state levels of every exon-intron junction in wild-type, PTC-containing, and missense-containing precursor mRNAs of both the nonrearranging dihydrofolate reductase (DHFR) and the somatically rearranging Ig- micro genes. We find that each exon-intron junction's abundance and, therefore, the rate of intron removal, is not significantly affected by the presence of a PTC in a neighboring exon in either the DHFR or Ig- micro pre-mRNA. Similarly, the abundance of the uncleaved Ig- micro polyadenylation sites does not differ between wild-type and PTC-containing Ig- micro pre-mRNAs. Our Ig- micro data were confirmed by RNase protection analyses, and multiple cell isolates were examined to resolve differences with previously published data on steady-state pre-mRNA levels. We conclude that the presence of a PTC affects the rate of neither splicing nor the cleavage step of 3' end formation during pre-mRNA processing in the nucleus. Our results are discussed with respect to existing evidence for nuclear surveillance mechanisms.

Parallel island genetic algorithm applied to a nuclear power plant auxiliary feedwater system surveillance tests policy optimization

In this work, we focus the application of an Island Genetic Algorithm (IGA), a coarse-grained parallel genetic algorithm (PGA) model, to a Nuclear Power Plant (NPP) Auxiliary Feedwater System (AFWS) surveillance tests policy optimization. Here, the main objective is to outline, by means of comparisons, the advantages of the IGA over the simple (non-parallel) genetic algorithm (GA), which has been successfully applied in the solution of such kind of problem. The goal of the optimization is to maximize the system's average availability for a given period of time, considering realistic features such as: i) aging effects on standby components during the tests; ii) revealing failures in the tests implies on corrective maintenance, increasing outage times; iii) components have distinct test parameters (outage time, aging factors, etc.) and iv) tests are not necessarily periodic. In our experiments, which were made in a cluster comprised by 8 1-GHz personal computers, we could clearly observe gains not only in the computational time, which reduced linearly with the number of computers, but in the optimization outcome.

Nuclear incident could prompt more costly inspections [power plants

On 7 May 2002, the U.S. Nuclear Regulatory Commission (NRC, Rockville, Md.) formally heard the operator of an Ohio reactor report on the causes of a situation that could very nearly have led to a serious loss-of-coolant accident. The situation is now fairly well understood and can be considered isolated, but questions remain as to whether nuclear surveillance both by the NRC and plant operators is as rigorous as it needs to be.

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.

Insider and Outsider Perspectives in the Anthropology of Science: A Cautionary Tale

Understanding the categories in terms of which people act is a basic tenet of anthropological research. This principle should apply to the study of scientists, yet analysts (social scientists and others) often do not address the content of science and, therefore, ignore a significant aspect of the conceptual framework within which scientists act. This paper examines, in the case of the development of the American secret nuclear surveillance system, the limitations of interpretations that do not adequately analyze such a conceptual framework. It also examines the implications of this case for an anthropology of science.

Assessment of the ductile-to-brittle transition region with 1X-WOL specimens

Several of the nuclear reactor surveillance programs that were started in the early 1970s incorporated some 1X-WOL type fracture mechanics specimens. The 1X-WOL is an early specimen design intended specifically to obtain valid K Ic values on highly embrittled materials. This specimen is now known to be unsuitable when the material being tested has sufficient fracture toughness to develop elastic-plastic performance. The objective of this project was to evaluate modifications that can be easily made by machining within the hot cell to make the specimens viable for evaluation in the ductileto-brittle transition region using elastic-plastic data development and analyses. Three modifications were tried: (1) remove 7.4 mm from the back face to make the compact specimen H/W ratio of 0.6; (2) a side groove of 25% to decrease the initial remaining ligament strength; and (3) combining side grooving and back face removal, both in lesser amounts, to weaken the remaining ligament strength. An A 533 grade B steel that had already been well documented in toughness properties was selected for this work. The first modification did not work because the remaining ligament was made too small to sufficiently control loss of constraint. The second and third modifications were successful from the standpoint of reproducing the results of the previous material characterization. A recommendation is made for the adoption of the second modification since it is more easily performed as a machining operation in the hot cell.

Atom probe characterization of the microstructure of nuclear pressure vessel surveillance materials after neutron irradiation and after annealing treatments

Microstructural changes due to neutron irradiation of weld and forging materials were characterized using the atom probe field ion microscope (APFIM). Neutron-induced clusters containing Cu, P, Ni, Mn and Si were detected in the high copper weld (0.24 at.% Cu) after irradiation to fluences of 6.6 × 10 22 and 3.47 × 10 23 n m −2; only phosphorus atmospheres were observed in the low copper forging material (0.02 at.% Cu) irradiated to an intermediate fluence of 1.5 × 10 23 n m −2. These results are in agreement with previous studies and with their respective measured transition temperature shifts. In addition, APFIM experiments were carried out on the high fluence weld material after two post-irradiation annealing treatments. The first annealing treatment of 168 h at 454°C is similar to the proposed condition for in situ pressure vessel annealing and the second, 29 h at 610°C, is similar to the final stress relief heat treatment employed in vessel fabrication. Annealing at 454°C led to coarsening of the copper-enriched precipitates and a 92% recovery of the radiation-induced transition temperature shift. Essentially complete rehomogenization of the solutes was obtained in the simulated stress relief treatment at 610°C.

Reviews of Books

Reviews of Books

Spying without Spies: Origins of America’s Secret Nuclear Surveillance System by Charles A. Ziegler, David Jacobson

TECHNOLOGY AND CULTURE Book Reviews 391 Spying without Spies: Origins ofAmerica’s Secret Nuclear Surveillance Sys­ tem. By Charles A. Ziegler and David Jacobson. Westport, Conn., and London: Praeger, 1995. Pp. xi+242; notes, bibliography, in­ dex. $49.95. Now that the age of heroic inventors is long over, many people believe that technology has a life of its own. This book disproves that idea, for it clearly demonstrates that technological advances come neither from brilliant inventors nor from a mysterious and autono­ mous force, but out of committee meetings. The technology in question is the detection of nuclear explosions from afar, more specificallythe techniquesusedbythe UnitedStatesto detect the first Soviet atomic bomb, code-namedJoe-1. Interestingly, from the time of Hiroshima on, the press and the American public believed that detecting an atomic explosion, even in a remote part of the Soviet Union, would be a simple matter. After all, wouldn’t so powerful an explosion produce tremors in the earth that seismo­ graphs could register and create enough radioactive dust to set off Geiger counters around the world? In fact, as the authors make abun­ dantly clear, detecting an atomic explosion was an extraordinarily costly and complex undertaking, involving dozens ofairplanes flying around the clock, several highly specialized laboratories, and hun­ dreds of aviators, technicians, and scientists to staff them. Thanks to recently declassified documents in the National Ar­ chives and to diligent searching on the authors’ part, the technical and scientific complexities ofthe detection program are now becom­ ing clear. Sections of this book deal with the techniques of meteoro­ logical surveying, air filtration, radiochemistry, and seismology, as well as various attempts to detect high-altitude air pressure waves. Science and technology are not the main theme of the book, how­ ever, and they occupy only three of the eleven chapters. Rather, it is the rivalries among the army, the navy, the army air force, the Atomic Energy Commission, and the Central Intelligence Group or Agency that take center stage. Hidden behind a profusion of acro­ nyms (AFMSW-1, AFOAT-1, AFTAC) and code words (Fitzwilliam, Crossroads, Murray Hill Area, etc.), these organizations tried as hard to preempt each other in controlling a lucrative new activity as to detect a Soviet bomb. This is the story of the bureaucratic turfwars, the endless committee meetings, and the intricate review procedures that the detection program generated. This emphasis is a result of the sources, consisting of declassified memos, letters, and reports, most of them written as ammunition in the bureaucratic guerrilla wars. A reader interested in the results of the program will wonder how anything ever got done, with so many bureaucrats (in and out of uniform) squabbling, conniving, and keeping each other in the dark with elaborate and counterproductive security measures. In fact, the detection program almost did not happen. Although 392 Book Reviews TECHNOLOGY AND CULTURE the press, the public, and lower-level officials expected a Soviet bomb to explode imminently, the authors show that the most power­ ful and knowledgeable officials in the nuclear establishment—Gen­ eral Leslie Groves and J. Robert Oppenheimer, and later James B. Conant and Vannevar Bush—were quite certain that the United States could enjoy its nuclear monopoly for a decade or longer. They based their rosy assessment on the conviction that atomic bombs could only be made out of the high-grade uranium found in a single mine in the Belgian Congo, a rare substance for which the United States had cornered the market. They also believed that the Russians were technologically and culturally too backward to catch up with the United States or to develop methods to use low-grade uranium ores. As a result, they allowed the armed services to develop their detection methods at a leisurely pace without guidance from above or a sense of urgency. When the Soviets exploded Joe-1 barely four years after Hiroshima, “Top Washington officials . . . wire shocked and surprised” (p. 212). This is a fine, well-researched book that will delight historians of American bureaucracy and the military. Historians of technology will find several interesting sections in it, but they will wish for more. Daniel R. Headrick Dr. Headrick, professor of history and...