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.