Figure 2.

DDX55 crosslinks to helices H62-63 of the 28S ribosomal RNA sequence in vivo. (A) HEK293 cells expressing DDX55-Flag or the Flag tag alone were crosslinked using light at 254 nm. The tagged proteins and crosslinked RNAs were retrieved on α-Flag beads under native conditions and subjected to a partial RNase digest. Complexes were then immobilized on NiNTA in denaturing conditions and co-purified RNA fragments were [³²P] labelled, ligated to sequencing adaptors. After elution, protein-RNA complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane and radiolabelled RNAs were detected by autoradiography. (B) The regions of the membranes containing crosslinked DDX55-Flag complexes were excised, as well as a corresponding region of the lane containing the Flag sample. RNAs were released by proteinase treatment, purified and converted to a cDNA library that was subjected to deep sequencing. The obtained sequence reads were mapped to the human genome and the relative proportions of reads mapped to gene features encoding different classes of RNA was determined. Abbreviations – ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), microRNA (miRNA), mitochondrial RNA (mtRNA). (C) The number of reads mapping to each nucleotide of the rDNA sequence encoding the 47S pre-rRNA is shown above a schematic view of the transcript (upper panel). The number of mutations, arising during reverse transcription due to the presence of amino acid-crosslinked nucleotides, mapping to each nucleotide is also shown (lower panel). (D) The number of sequencing reads in the DDX55-His₆-Prc-Flag CRAC data mapping to each nucleotide of the 28S rRNA sequence is shown on the secondary structure of the mature 28S rRNA using the indicated colour scale. A magnified view of the region crosslinked by DDX55 is shown. (E) The number of sequencing reads in the DDX55-His₆-Prc-Flag CRAC data mapping to each nucleotide of the tertiary structure of the 28S rRNA (grey) within the mature LSU (PBD: 4V6X) is shown using a colour scale as in (D). The ribosomal proteins of the LSU are shown in surface view in pale cyan. The positions of key ribosomal features are indicated. CP – central protuberance