Abstract
Ribosomal RNAs are processed in a complex pathway. We profiled rRNA processing intermediates in yeast at single-molecule and single-nucleotide levels with circularization, targeted amplification and deep sequencing (CircTA-seq), gaining significant mechanistic insights into rRNA processing and surveillance. The long form of the 5' end of 5.8S rRNA is converted to the short form and represents an intermediate of a unified processing pathway. The initial 3' end processing of 5.8S rRNA involves trimming by Rex1 and Rex2 and Trf4-mediated polyadenylation. The 3' end of 25S rRNA is formed by sequential digestion by four Rex proteins. Intermediates with an extended A1 site are generated during 5' degradation of aberrant 18S rRNA precursors. We determined precise polyadenylation profiles for pre-rRNAs and show that the degradation efficiency of polyadenylated 20S pre-rRNA critically depends on poly(A) lengths and degradation intermediates released from the exosome are often extensively re-polyadenylated.