Separate structural elements within internal transcribed spacer 1 ofSaccharomyces cerevisiaeprecursor ribosomal RNA direct the formation of 17S and 26S rRNA

Abstract

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.

An ITS phylogeny ofLeccinumand an analysis of the evolution of minisatellite-like sequences within ITS1

“…The length variation observed in the ITS1 spacer in most species of Leccinum examined by us is caused by an insert varying in length from 240 to 749 bp. According to Van Nues et al (1994) the ITS1 spacer in yeasts is organised in two functionally and structurally distinct halves with highly conserved nucleotide regions at three different positions. In relation to the position of the A2 processing site, we assume the insert in the ITS1 spacer in Leccinum is situated in domain III ( see FIG.…”

“…They also show that these non-functional ITS regions are characterized by the presence of more mutations, especially in the more conserved regions. According to Van Nues et al (1994) deletions in domain III severely reduce the formation of 17S rRNA. Given the fact that, in those samples in which we found the insert, no other sequences lacking the insert were present, we have to conclude the ITS1 region is likely to be still functional.…”

“…To facilitate the alignment, the minisatellite in Leccinum was left out. Conserved regions as indicated by Van Nues et al (1994) were found by comparison of primary and secondary structures with yeast sequences using secondary structure predictions performed in RNAdraw version 1.1 (Matzura and Wennberg 1996).…”

Domain II Hairpin Structure in ITS1 Sequences as an Aid in Differentiating Recently Evolved Animal and Plant Pathogenic Fungi

“…It is an essential region and removal of the various domains has different effects on the production of the rRNA subunits [9]. The current model for rRNA formation involves the transcription of the complete rRNA gene cluster as a single unit and subsequent folding of the RNA prior to translation (see [10]).…”

“…The current model for rRNA formation involves the transcription of the complete rRNA gene cluster as a single unit and subsequent folding of the RNA prior to translation (see [10]). Particular structures in the ITS transcript, such as helices and loops, are believed to play a role in anchoring and folding the complete RNA molecule, and to act as markers to ensure their exclusion from the final rRNA-protein complexes [9,11].…”