Characterization and localization of the RNA synthesized in mature avian erythrocytes

Abstract

Experimental Cell Research 96 (1975) RNA present and synthesized in mature hen erythrocytes in vivo and in vitro has been studied with electron microscopic , autoradiographic , and biochemical methods. The average hen erythrocyte contains a minimum of 0.02-0.04 pg RN A which is predominantly, if not exclusively, located in the nucleus. The incorporation of[3HJuridine into this RNA is very low but is clearly demonstrable in vivo and in vitro using different incubation media. The specific efficiencies of the incorporation assays are compared. The incorporated radioactivity is contained in RN A as shown by its selective sensitivity to hydrolysis in mild alkali and with ribonuclease a nd is not hybridized to DNA as shown by Cs.SO" gradient centrifugation of total hen erythrocyte nucleic acids. LiCI precipitation as well as gel electrophoresis on agarose under denaturing and non-denaturing conditions has revealed that this labelled RNA is of high-molecular weight a nd covers a broad range from ca 0.25 to 5.0 million D, with gel-electrophoretically detectable peaks at positions corresponding to 1.8, 1.0 and 0.5 million D. The pattern obtained is virtually identical after the different incorporation condi tion s and was also sim il ar to that obtained in reticulocyte cells of animals made anaemic with phenylhydrazine. The incorporation is inhibited by actinomycin D with a sensitivity characteristic for mRN A formation a nd is also reduced in the presence of a-amanitin. The labelled RNA remains within th e confinements of the nucleus and does not seem to be transported into the cytoplasm. It appears to be preferentially enriched in the interchromatinic channels . It is concluded that mature hen erythrocytes are capable of transcribing a small part of their genome into pre-mRNA at a very reduced level but that this RNA is not only excluded from functioning in tra nslation but also from nucleocytoplasmic transport. The findings are discussed in relation to the controversial literatu re in this field, a nd possible functions of thi s RNA are hypothesized.Various forms of cell differentiation are characterized by a progressive cessation of the sy nthesis of most, if not all, classes of RNA. In some late stages of such differentiation processes one even notes a general breakdown of RNA-containing structures and the mature cell is almost emptied of RNA . The formation of the red blood cell, in particular in animals with nucleated erythrocytes , has become of special importance in studie s of the programmed switch-off sequence of the transcription of most, and finally perhaps of all, genes. It has been shown that during erythropoiesis the nuclear content and the nuclear envelope is greatly reduced , the chromatin condenses and contains an additional special histone , the nucleolus disappears, concomitant with the cessation of ribosome formation , the number of transcribed genes decreases , leaving the transcription of one class of genes in the Exprl Cell Res 96 (1975)

Changes of nucleosome frequency in nucleolar and non-nucleolar chromatin as a function of transcription: an electron microscopic study

“…The beaded pattern of the inactivated nucleolar chromatin is indistinguishable from that of the almost completely inactive chromatin of hen erythrocyte nuclei (Zentgraf , Scheer and Franke, 1975) that was spread under identical conditions ( Figure 5a). Nucleosomes, however, are similar in size and staining properties to RNA polymerase molecules (for references, see Franke et al, 1976a) .…”

A constitutive nucleolar protein identified as a member of the nucleoplasmin family.

“…The latter have been reported to be inactive in nucleolar transcription and lack RNA polymerase I (e.g. MacLean et al, 1973;Zentgraf et al, 1975;Kruger and Seifart, 1977;Hentschel and Tata, 1978;MacLean and Gregory, 1981).…”

Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody

“…Such dots seem to correspond to the 'residual nucleolar structure' seen in electron micrographs of erythrocytes of amphibia (fig. 7 e; cf [37]) and birds [37][38][39][40] which have been reported to be inactive in rDNA transcription and to lack RNA polymerase I [40][41][42][43].…”