Synthesis of pre-rRNA and mRNA is directed to a chromatin-poor compartment in the macronucleus of the spirotrichous ciliate Stylonychia lemnae

Abstract

In contrast to the chromosomal genome organization common to most eukaryotes, DNA in the macronucleus of spirotrichous ciliates like Stylonychia lemnae is organized into small gene-sized nanochromosomes. We intended to elucidate whether a spatial organization of nucleoli similar to other eukaryotes can be found in absence of typical chromosomes. Whereas micronuclei of Stylonychia exhibit homogenously stained heterochromatin and possess no nucleoli, macronuclear chromatin is compartmentalized and contains numerous putative nucleoli. Since the identity of these spherical structures has never been unequivocally demonstrated to date, we applied immunofluorescence techniques together with confocal laser scanning microscopy to identify nucleolar bodies in the macronucleus of Stylonychia and to analyse their spatial organization. We found that multiple spherical bodies, which fulfil nucleolar function, occupy a peripheral localization in mature macronuclei. Using fibrillarin/Nop1p as a nucleolar marker, we monitored the assembly of such nucleolar bodies during macronuclear differentiation. 3D-FISH experiments revealed that rRNA genes are mostly concentrated adjacent to but not inside of fibrillarin/Nop1p-containing bodies. We further showed that transcription sites for rRNA synthesis but also for mRNA synthesis occur predominantly at surfaces of nucleolar bodies and chromatin-poor spaces bordering condensed chromatin. Our data suggest that transcription of rRNA genes in the macronucleus of Stylonychia does not rely on a classical nucleolus-type organization. We assume that vectorial synthesis and processing of rRNA and mRNA is directed to a functional interchromatin compartment.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora)

“…Our results are in a good agreement with the fi ndings of Postberg et al (2006) in the macronucleus of Stylonychia lemnae, which displays gene-sized mini-chromosomes. In this ciliate rDNAs are found to occur adjacent to, but outside the nucleoli.…”

“…Thus transcription, or at least its initiation, should start within the outer domains of condensed chromatin and the subsequent processing of rRNA should occur in the interior of nucleolar domains. It was assumed that the absence of typical chromosomes determines this alternative spatial organization of the nucleolus (Postberg et al, 2006). Our data show that nucleoli within macronuclei with subchromosomal DNAs (D. nasutum) can also conduct rRNA processing in thr opposite direction compared to the "classical" nucleoli of Metazoa.…”

Localization of fibrillarin in the nucleoli of ciliate species with subchromosomal organization of macronuclear genome

“…The authors assumed that the absence of typical chromosomes could determine such alternative spatial organization as observed in S. lemnae nucleoli (Postberg et al, 2006). However, our data indicate that the small size and the absence of typical chromosomes cannot be the only factors determining the inverted spatial organization of some ciliate nucleoli.…”

“…The nucleoli with such "inverted" direction of vectorial processing of rRNA transcripts were observed by Postberg et al (2006) in the S. lemnae nucleolar bodies. The authors assumed that the absence of typical chromosomes could determine such alternative spatial organization as observed in S. lemnae nucleoli (Postberg et al, 2006).…”

“…Each of these DNA molecules is terminated by telomeres at both ends, lacks a centromere and is usually considered as a nano-, or mini-chromosome (Zagulski et al, 2004;Pimenov et al, 2006;Jung et al, 2011). Postberg et al (2006) showed that spherical nuclear structures, which fulfil nucleolar function in the gene-sized ciliate Stylonychia lemnae, do not represent true nucleoli in a classical use of the term, because processing of rRNA occur in a bipartite and inverse manner compared to typical metazoan nucleolar organization. Postberg et al (2006) assumed that the absence of typical chromosomes could determine an alternative spatial organization of S. lemnae nucleolar bodies.…”