Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins

AbstractMammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase μ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

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New Markers for Separating Benign From Malignant Mesothelial Proliferations: Are We There Yet?

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2015-0240-sa ◽

2015 ◽

Vol 140 (4) ◽

pp. 318-321 ◽

Cited By ~ 83

Author(s):

Andrew Churg ◽

Brandon S. Sheffield ◽

Francoise Galateau-Salle

Keyword(s):

Messenger Rna ◽

Glucose Transporter ◽

Epithelial Membrane Antigen ◽

Rna Binding ◽

Individual Case ◽

Homozygous Deletion ◽

Major Drawback ◽

Glucose Transporter 1 ◽

Limited Sensitivity

The separation of benign from malignant mesothelial proliferations is crucial to patient care but is frequently morphologically difficult.Context.— To briefly review adjunctive tests claimed to be useful in this setting and to examine in detail 2 new tests: p16 fluorescence in situ hybridization (FISH) and BRCA1-associated protein 1 (BAP1) immunohistochemistry.Objective.— Literature review with emphasis on p16 FISH and BAP1 immunohistochemistry.Design.— Glucose transporter-1, p53, insulin-like growth factor 2 messenger RNA–binding protein 3 (IMP-3), desmin, and epithelial membrane antigen have all been claimed to mark either benign or malignant mesothelial processes, but in practice they at best provide statistical differences in large series of cases, without being useful in an individual case. Homozygous deletion of p16 by FISH or loss of BAP1 has only been reported in malignant mesotheliomas and not in benign mesothelial proliferations. BAP1 appears to be lost more frequently in epithelial than mixed or sarcomatous mesotheliomas. Homozygous deletion of p16 by FISH is seen in pleural epithelial, mixed, and sarcomatous mesotheliomas, but it is much less frequent in peritoneal mesothelioma. The major drawback to both these tests is limited sensitivity; moreover, failure to find p16 deletion or BAP1 loss does not make a mesothelial process benign.Results.— In the context of a mesothelial proliferation, the finding of homozygous deletion of p16 by FISH or loss of BAP1 by immunohistochemistry is, thus far, 100% specific for malignant mesothelioma. The limited sensitivity of each test may be improved to some extent by running both tests.Conclusions.—

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Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.211 ◽

1999 ◽

Vol 10 (1) ◽

pp. 211-223 ◽

Cited By ~ 164

Author(s):

Dusan Cmarko ◽

Pernette J. Verschure ◽

Terence E. Martin ◽

Michael E. Dahmus ◽

Sabine Krause ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Ultrastructural Level ◽

Structural Domain ◽

Mrna Transcription ◽

Rna Transcription ◽

Core Proteins ◽

Transcription Sites ◽

Nascent Transcripts

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

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Clinical Implications of Current Developments in Genitourinary Pathology

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2012-0210-ra ◽

2013 ◽

Vol 137 (7) ◽

pp. 887-893 ◽

Cited By ~ 6

Author(s):

Amy G. Zhou ◽

Christopher L. Owens ◽

Ediz F. Cosar ◽

Zhong Jiang

Keyword(s):

Messenger Rna ◽

Rna Binding ◽

Prognostic Biomarker ◽

Rna Binding Protein ◽

Clinical Implications ◽

Factor Ii ◽

Personal Experiences ◽

Hybridization Test ◽

Genitourinary Cancers

Context.—Several developments in genitourinary pathology are likely to change our understanding and management of some genitourinary cancers considerably. Objective.—To review 5 stories in genitourinary pathology: (1) fusion in the ETS (E26) gene family in prostatic adenocarcinoma; (2) insulin-like growth factor II messenger RNA-binding protein 3 (IMP3), an important prognostic biomarker for kidney and bladder cancers; (3) translocation renal cell carcinoma; (4) UroVysion fluorescence in situ hybridization test in urine cytology for detection of bladder cancer; and (5) the use of triple immunostaining for diagnosis of prostate cancer. Data Sources.—Literature review and authors' personal experiences. Conclusions.—Many scientific findings have contributed recently to the understanding of the natural pathogenesis and progression of genitourinary cancers. This translational research helps in diagnosing, predicting, and potentially, treating genitourinary cancers.

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Rotary Beveling for In Situ Thin-Section Microscopy of Cell Cultures

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099192 ◽

1981 ◽

Vol 39 ◽

pp. 550-551

Author(s):

Dean A. Handley ◽

Jack T. Alexander ◽

Shu Chien

Keyword(s):

Cell Growth ◽

Cell Cultures ◽

Molecular Interactions ◽

Convenient Method ◽

Petri Dish ◽

Simple Method ◽

Thin Section Microscopy ◽

Thin Sectioning ◽

Organic Fluids

In situ preparation of cell cultures for ultrastructural investigations is a convenient method by which fixation, dehydration and embedment are carried out in the culture petri dish. The in situ method offers the advantage of preserving the native orientation of cell-cell interactions, junctional regions and overlapping configurations. In order to section after embedment, the petri dish is usually separated from the polymerized resin by either differential cryo-contraction or solvation in organic fluids. The remaining resin block must be re-embedded before sectioning. Although removal of the petri dish may not disrupt the native cellular geometry, it does sacrifice what is now recognized as an important characteristic of cell growth: cell-substratum molecular interactions. To preserve the topographic cell-substratum relationship, we developed a simple method of tapered rotary beveling to reduce the petri dish thickness to a dimension suitable for direct thin sectioning.

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High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166944 ◽

1996 ◽

Vol 54 ◽

pp. 896-897

Author(s):

G. W. Hacker ◽

I. Zehbe ◽

J. Hainfeld ◽

A.-H. Graf ◽

C. Hauser-Kronberger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Messenger Rna ◽

High Performance ◽

Detection System ◽

Direct Methods ◽

Genetic Alterations ◽

Chain Reaction ◽

Protein Encoding ◽

Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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