Fine Structural Specific Visualization of RNA on Ultrathin Sections

Biggiogera, Marco; Fakan, Stanislav

doi:10.1177/002215549804600313

Cited by 55 publications

(40 citation statements)

References 13 publications

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“…Immunolabelled ultrathin sections were stained either with the EDTA method (Bernhard, 1969) to visualize RNP constituents, or with terbium citrate (Biggiogera and Fakan, 1998;Trentani et al, 2003) for selective RNA staining; Epon sections were conventionally stained with uranyl-lead. All samples were observed in a Zeiss EM 900 electron microscope operating at 80 kV.The gold grain contrast was digitally enchanced on the scanned micrographs using Adobe Photoshop.…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural analysis of testes from mice fed on genetically modified soybean

Vecchio¹,

Cisterna²,

Malatesta³

et al. 2009

Eur J Histochem

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We have considered the possible effects of a diet containing genetically modified (GM) soybean on mouse testis. This organ, in fact, is a well known bioindicator and it has already been utilized, for instance, to monitor pollution by heavy metals. In this preliminary study, we have focussed our attention on Sertoli cells, spermatogonia and spermatocytes by means of immunoelectron microscopy. Our results point out that the immunolabelling for Sm antigen, hnRNPs, SC35 and RNA Polymerase II is decreased in 2 and 5 month-old GM-fed mice, and is restored to normal at 8 months. In GM-fed mice of all ages considered, the number of perichromatin granules is higher and the nuclear pore density lower. Moreover, we found enlargements in the smooth endoplasmic reticulum in GM-fed mice Sertoli cells. A possible role played by traces of the herbicide to which the soybean is resistant is discussed.

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Section: Methodsmentioning

confidence: 99%

Ultrastructural analysis of testes from mice fed on genetically modified soybean

Vecchio¹,

Cisterna²,

Malatesta³

et al. 2009

Eur J Histochem

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“…Finally, the distribution of CF I m and SC35 was determined in HeLa cells that had been stained with terbium. Terbium staining can be used to specifically visualize RNA-containing nuclear compartments (Biggiogera and Fakan, 1998). As shown in Figure 6C, gold labeling for CF I m colocalized with SC35 over terbium-stained RNA fibrils.…”

Section: Cf I M 68 Localization Is Dependent On Ongoing Transcriptionmentioning

confidence: 97%

“…All the grids were rinsed with PBS and distilled water and finally stained with the EDTA regressive technique of Bernhard (1969) preferential for RNP-containing nuclear components. Some grids with the sections were floated onto 0.2 M terbium citrate, prepared according to Biggiogera and Fakan (1998), for 1 h at room temperature, and then rapidly rinsed with water and dried. Stained specimens were observed with a Zeiss EM900 electron microscope equipped with a 30-m objective aperture and operating at 80 kV.…”

Section: Immunoelectron Microscopymentioning

confidence: 99%

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

Cardinale

Cisterna

Bonetti

et al. 2007

MBoC

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Mammalian cleavage factor I (CF I m ) is an essential factor that is required for the first step in pre-mRNA 3 end processing. Here, we characterize CF I m 68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF I m 68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF I m 68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF I m 68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFI m 68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF I m 68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3 end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF I m 68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus. INTRODUCTIONMost functional mRNAs of eukaryotic genes are generated from their primary transcripts (pre-mRNAs) through RNA splicing and 3Ј end polyadenylation. Removal of introns occurs in the spliceosome, a complex composed of five small ribonucleoprotein particles (U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles [snRNPs]) and many non-snRNPs splicing factors, including members of the arginine-serine (SR) family of proteins. The mature 3Ј ends of mRNAs are generated by endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream cleavage product. Biochemical studies have identified six factors required for efficient processing in vitro: the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), and two cleavage factors, mammalian cleavage factor I m [CF I m ] and CF II m , are necessary for the cleavage reaction. Polyadenylation requires in addition to CPSF, poly(A) polymerase, and the nuclear poly(A) binding protein 1 (PABPN1, previously called PAB II, for review, see Wahle and Rü egsegger, 1999). Other proteins involved in either transcription, such as the carboxy-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (Flaherty et al., 1997;Hirose and Manley, 1998;Millevoi et al., 2002).The mammalian cell nucleus is a highly structured and dynamic compartment. Many nuclear factors are localized in morphologically well-defined structural units that include the nucleolus and several "nuclear bodies," such as the Cajal ...

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“…FACS diagrams confirmed a significant degree of yellow staining (vertical axis). On the other hand, a more variable intensity of the green stain (DNA, horizontal axis) was indicative of a less compact chromatin structure.RNA containing structures can be identified in the electron microscope by the EDTA regressive staining technique, based on the chelation of uranyl ions by neutral EDTA 18,19 . EDTA regressive staining in an Epon section shows densely contrasted structures corresponding to ribonucleoprotein constituents, while DNA containing structures appear greyish or bleached (see control spermatocyte sections in Fig.…”

mentioning

confidence: 99%

RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse

Rassoulzadegan

Grandjean

Gounon

et al. 2006

Nature

831

643

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3 Paramutation, first observed in maize 1 and subsequently in a variety of plants 2 , is a heritable epigenetic change of the phenotype of a "paramutable" allele, initiated by interaction in heterozygotes with a "paramutagenic" form of the locus. Often referred to as an exception to the law of Mendel, which states that genetic factors segregate unchanged from heterozygotes, paramutation is meiotically stable and inherited in the absence of the inducing allele. To date, the closest observations in an animal species were changes in the DNA methylation profiles directed by the allelic locus in the mouse that we and others described as "transvection" or "paramutation-like" effects 3,4 . We now report a modification in the phenotypic expression of the wild type allele of the Kit receptor gene in the progeny of heterozygotes with a null insertion mutant. The "paramutated (Kit*)", genotypically wild type animals, maintain the white-spotted phenotype characteristic of Kit mutants in the absence of the mutant allele. The efficient paternal and maternal inheritance of the paramutated state raises the question of a possible molecular support of the epigenetic information. Non-Mendelian phenotype distributionThe tm1Alf mutation (MGI:2449782, initially designated Kit W-LacZ ) was engineered 5 by inserting a 3 kb Neo-LacZ cassette downstream of the initiator ATG. A unique mRNA of the same size with the ß-galactosidase coding sequence is expressed under control of the Kit promoter and regulatory sequences. The mutation abrogates the synthesis of the Kit tyrosine kinase receptor, which plays a critical role in several developmental processes including germinal differentiation, hematopoiesis and melanogenesis. Accordingly, Kit tm1Alf homozygotes die shortly after birth and heterozygotes show a white tail tip and white feet (Fig. 1). We initially observed an abnormal segregation of phenotypes in the progeny of crosses between two heterozygous parents. Wild type genotypes were identified by the absence of both LacZ sequences determined by genomic PCR analysis and ß-galactosidase expression by in situ X-Gal staining (not shown), and further confirmed by Southern blot analysis (Fig. 1c).However, it was striking that most of these genetically Kit +/+ mice maintained the white patches characteristic of the parental heterozygotes ( Fig. 1 and Table 1). The occurrence of this modified, "paramutated" form of the Kit + allele (Kit* phenotype) was not restricted to the progeny of heterozygote intercrossing, but also observed in Kit tm1Alf / + crosses with wild type partners, 4 independently of the gender combination (Table 1). It was not dependent on the genetic background of the mice since the same phenotypes were found with the original 129/Sv Kit tm1Alf / + heterozygotes and after at least 6 generations of back-crosses of the mutation onto the C57Bl/6 and B6D2 genetic backgrounds (Supplementary Table 1). The paramutated phenotype was inherited, with a variable phenotypic extent depending on the crosses (Supplementary Fig. 1). It was mos...

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Fine Structural Specific Visualization of RNA on Ultrathin Sections

Cited by 55 publications

References 13 publications

Ultrastructural analysis of testes from mice fed on genetically modified soybean

Ultrastructural analysis of testes from mice fed on genetically modified soybean

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse

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