(A) Processing of the precursor ribosomal RNA (rRNA) to the four mature rRNAs. Blue boxes indicate large subunit rRNAs (60S SU) and red boxes mark the small subunit rRNA (40S SU). The 35S precursor rRNA still harbors the two flanking regions 5′ETS and 3′ETS (external transcribed spacers) and the two internal transcribed spacer elements ITS1 and 2. Endonucleolytic cleavage sites (▾) and regions removed by exonucleolytic trimming (→) are labeled. Enzymes in italics indicate candidate proteins involved in processing or when several enzymes were described to act at the same site. For processing sites with question marks, no enzyme was identified until now. (NU) Nucleolus, (NP) nucleoplasm, (CP), cytoplasm. (B) Experimental design of the Riboprobing workflow. At first, an RNA linker is ligated to isolated RNA. Depending on whether the 3′ or the 5′ end is investigated, a different primer serves as the starting point of the reverse transcription. For the 5′ end, a site-specific primer is chosen (e.g., the C1C2 primer that allows discrimination of the different 27S pre-rRNA species). For the 3′ ends, the primer hybridizing to the linker (linker primer) is used to initiate cDNA synthesis. In the next step, the cDNA is amplified. For the 5′ ends, the primer used for cDNA synthesis is combined with a primer hybridizing to the linker. To investigate the 3′ ends, the linker primer is combined with a primer defining the region of interest, in this scheme the 3′ end of the 18S rRNA. Amplified DNA fragments are separated in agarose gels to deduce which pre-rRNA species were present in the sample.
