Figure 5.
NIFK regulates 28S and 5.8S rRNA maturation through processing of ITS1 site 2. (A) Schematic representation of human rRNA transcripts. Upper, human 47S precursor rRNA showing the transcribed spacers (ETS and ITS), coding sequences, and cleavage sites. Lower left, illustration of 47S pre-rRNA processing pathways. The alternative steps are colored gray. Lower right, overview of pre-rRNA intermediates. Arrowheads indicate the positions of the probes used in Northern blot analysis. (B-F) Northern blot analyses showing the pre-rRNAs derived from siNIFK transfected U2OS cells. The specific rRNA species were detected using probes complementary to the regions downstream of site A0 of 5′ETS (P1, shown in B), between 18S and site 2a (P2, shown in C), between site 2a and 2 of ITS1 (P3, shown in D), between 5.8S and site 4 of ITS2 (P4, shown in E), and 18S, 5.8S, and 28S rRNA (probes 5, 6 and 7, shown in F). (G) Northern blot analysis of the pre-rRNAs derived from phenotypic rescued cells using probe P4. The complementation of NIFK silencing by NIFK and NIFK-KF mutant following that described in Fig. 4. (H) The same as (E), except that the siRPL5 transfected U2OS cells are also compared.