Functional analysis of transcribed spacers of yeast ribosomal DNA.

Abstract

Making use of an rDNA unit, containing oligonucleotide tags in both the 17S and 26S rRNA gene, we have analyzed the effect of various deletions in the External Transcribed Spacer (ETS) and in one of the Internal Transcribed Spacers 1 (ITS1) on the process of ribosome formation in yeast. By following the fate of the tagged transcripts of this rDNA unit in vivo by Northern hybridization we found that deleting various parts of the ETS prevents the accumulation of tagged 17S rRNA and its assembly into 40S subunits, but not the formation of 60S subunits. Deleting the central region of ITS1, including a processing site that is used in an early stage of the maturation process, was also found to prevent the accumulation of functional 49 S subunits, whereas no effect on the formation of 60S subunits was detected. The implications of these findings for yeast pre‐rRNA processing are discussed.

A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar

“…Although rDNA repeats are best known for the high degree of intraspecific homogeneity resulting from concerted evolution, another evolutionary mechanism involving ribosomal units has been described in eukaryotic genomics, where an 'nonconcerted' evolution can be a stable genetic condition in which some copies of a tandem repetitive gene are pseudogenes in addition to functional copies (Buckler et al, 1997). Van der Sande et al (1992) and Musters et al (1990) have demonstrated that both ITS regions play a primary role in rRNA processing. Deletions or mutations within ITS2, complete omission of ITS2 or replacement of ITS2 in Saccharomyces cerevisiae and in other yeasts result in failure to produce mature 5.8S and 26S rRNAs (Musters et al,1990;van der Sande et al, 1992;van Nues et al, 1995;Cote and Peculis, 2001).…”

“…Van der Sande et al (1992) and Musters et al (1990) have demonstrated that both ITS regions play a primary role in rRNA processing. Deletions or mutations within ITS2, complete omission of ITS2 or replacement of ITS2 in Saccharomyces cerevisiae and in other yeasts result in failure to produce mature 5.8S and 26S rRNAs (Musters et al,1990;van der Sande et al, 1992;van Nues et al, 1995;Cote and Peculis, 2001). The occurrence of mutations impairing the functional role of some ITS regions could act as a source of pseudogenes in addition to functional rDNA copies.…”

The 5′ end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.

“…Mutations in RATl were isolated as resulting in the nuclear accumulation of poly(A)+ RNA, as judged by immunofluorescence (Amberg et al, 1992 Di Segni et al, 1993;Kenna et al, 1993 Although a considerable number of trans-acting factors required for yeast pre-rRNA processing have been identified, little is known about the role of cis-acting sequences in the pre-rRNA. A system for the functional analysis of pre-rRNA sequences, using neutral tags inserted into the 18S and 25S rRNA regions, has been reported (Musters et al, 1989(Musters et al, , 1990 (Wittekind et al, 1988;Nogi et al, 1993), in combination with the conditional expression of mutant pre-rRNAs under the control of an RNA polymerase II GAL promoter (Nogi et al, 1991). This can be used to assess the effects of mutations in the prerRNA on growth and pre-rRNA processing.…”

Conserved secondary structures in the ITS2 of trematode pre‐rRNA

“…Although these transcribed spacer regions are obvious candidates for important roles in the control of ribosome biogenesis, elucidation of their biological function and of the molecular mechanisms involved in their accurate excision still remain a major challenge. Recent functional analyses performed on yeast S. cerevisiae ribosomal RNA genes clearly show that the structural integrity of the transcribed spacer regions is an essential prerequisite for correct processing of mature rRNA and biogenesis of active ribosomal subunits [3,4]. The derivation of reliable secondary structure models for each transcribed spacer region would undoubtedly represent a major step towards a Abbreviations: ITSZ, internal transcribed spacer 2.…”