Electron microscopic autoradiographic study of RNA synthesis in yeast nucleus

Abstract

SUMMARYHigh resolution autoradiography of isolated yeast nuclei has been used to investigate the distribution of newly formed RNA in the nucleus. The nuclei were isolated after incubation of spheromasts with 3H-uracil for 5 min. Most grains (80 %) are located over the dense crescent. In the dense crescent the grains seem to be associatedwith.the nucleolonema. An analysis of the nuclear RNA on polyacrylamide gels shows that the incorporated 3H-uracil is predominantly present in the initial (37s) ribosomal precursor RNA. The EM autoradiographic and biochemical data, taken together, strongly suggest that we have to ascribe a nucleolar function to the dense crescent of the yeast nucleus.When isolated yeast nuclei are observed by means of the phase-contrast microscope, a crescent-shaped segment of high optical density can be discerned [l]. EM studies have ascertained it to be very likely that this crescent is equivalent to the mammalian nucleolus. A large part of the "dense crescent" is occupied by a structure strongly resembling the nucleolonema of the mammalian nucleolus. Moreover, a correlated biochemical and electron microscopic study has shown that the dense crescent consists mainly of RNA and protein [2], The exponentially growing yeast cells from which the nuclei are isolated are endowed with a very high rate of synthesis of ribosomal RNA and a high ribosomes content [I]. Thus it is not surprising that yeast nuclei, compared with nuclei of animal cells, contain a relatively large amount of RNA which is mainly ribosomal precursor RNA [3]. EviExptl Cell Res 70 dence has come from several sources [3-51, that in yeast both species of ribosomal RNA derive from a common large precursor molecule. Hence it is clear that yeast, one of the most primitive eukaryotes, possesses a mechanism for the processing of ribosomal RNA which resembles that of higher eukaryotes. This raises the question whether, in yeast, as in higher eukaryotes, the ribosomal precursors will be confined to the region of the nucleus resembling a nucleolus.In order to obtain an insight into the intranuclear localization of the ribosomal precursor RNA, we have performed pulse labelling experiments with 3H-uracil followed by electron microscopic autoradiography. MATERIALS AND METHODS Organism and isotopeThe experiments were performed with spheroplasts [l] of the yeast strain Saccharomyces carlsbergensis,

mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

“…Considering that MTR13 is required for import and export and shuttles between cytoplasm and nucleus (Flach et al, 1994), it may form part of the putative bidirectional transporter, regulated by the nucleocytoplasmic GTPase cycle, which we have discussed above (see Figure 4). The recent report that a functional NLS is required both for protein import into the nucleus and for export of proteins from the nucleus (Guiochon-Mantel et al, 1994) The yeast nucleolus normally appears as a "gray" or "dense" crescent-shaped structure occupying more than one-third of the nuclear volume (Sillevis Smitt et al, 1972Smitt et al, , 1973. It lacks the obvious subcompartmentalization typically observed in nucleoli of higher eukaryotic cells and is much more extensively in contact with the nuclear envelope.…”

Yeast Nuclear Envelope Subdomains with Distinct Abilities to Resist Membrane Expansion

“…This is highlighted by the enrichment of certain DNA sequences, such as telomeres or late firing origins of replication, at the nuclear periphery (Heun et al, 2001;Hediger and Gasser, 2002). In addition, the nucleolus is limited to a crescent-shaped region, covering about one-third of the nuclear surface (Molenaar et al, 1970;Smitt et al, 1972). The nucleolus is a self-organizing structure that assembles by virtue of its specialized function in ribosome biogenesis (Dundr and Misteli, 2001).…”