Abstract
Previous work from our lab suggests that a group of interdependent assembly factors (A(3) factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A(3) factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA(3) pre-rRNA processing, even though the A(3) factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A(3) factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures.
Highlights
Previous work from our lab suggests that a group of interdependent assembly factors (A3 factors) is necessary to create early, stable preribosomes
Of yeast ribosomes requires ϳ180 trans-acting proteins termed assembly factors (AFs). The majority of these proteins are conserved across eukaryotes, are essential for cell growth, and are thought to function as scaffolding proteins, RNA chaperones, energy-consuming nucleoside triphosphatases (NTPases), nucleases, or posttranslational modifiers [3,4,5]
Purification of ribosome assembly intermediates allowed the identification of most AFs, and initial characterizations have shown in which steps of pre-rRNA processing many of these factors function
Summary
Previous work from our lab suggests that a group of interdependent assembly factors (A3 factors) is necessary to create early, stable preribosomes. In the mature 60S subunit, r-proteins L8 and L15 interact closely with each other as well as with the ITS2-proximal stem (helix 10), a conserved helix formed by base pairing between the 3= end of 5.8S rRNA and the 5= end of 25S rRNA (Fig. 1C) [29] These two rRNA ends are generated upon the removal of internal transcribed spacer 2 (ITS2) during processing of 27SB pre-rRNA (Fig. 1A). It was recently reported that Nop can be UV cross-linked within the ITS2-proximal stem, as well as a nearby stretch of 5.8S rRNA (helix 8) (Fig. 1C) [26] This result, as well as the known location of L8 and L15 in mature 60S subunits, suggests that the Pwp subcomplex binds within or near the ITS2-proximal stem and may play a role in folding rRNA and structuring this neighborhood
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