Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Fanconi anemia protein FANCI functions in ribosome biogenesis
Figure Lengend Snippet: FANCI functions in LSU pre-rRNA processing. ( A ). The 41S, 32S, and 12S pre-rRNAs are detected by a probe (P5) that hybridizes with ITS2. ( B ) FANCI depletion results in LSU pre-rRNA processing defects. HeLa cells were transfected with the indicated siRNAs, and total RNA was harvested after 72 h of depletion. Pre-rRNAs were separated by gel electrophoresis, and Northern blots were performed using radioactively labeled P5 probe. Northern blotting with a probe against the 7SL RNA was used as a loading control. Small illustrations of the pre-rRNAs are to the right of their respective bands. ( C – I ) profiles for Northern blotting from three biological replicates for mock ( C ), siFANCI-pool ( D ), siFANCI-2 ( E ), siFANCI-3 ( F ), siPES1 ( G ), siNOL11 ( H ), and siFANCD2 ( I ). Statistical significance was calculated using a two-way ANOVA with Sidak multiple comparisons test (mean ± SD). All comparisons are relative to siNT. The x axes of all graphs were equalized, except for siPES1 in G , which has larger RAMP values. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Unmarked bars are not significant. ( J ) FANCI depletion results in decreased mature 28S rRNA relative to 18S. The ratio of 28S/18S was calculated using an Agilent 2100 Bioanalyzer. Statistical significance was calculated using a one-tailed unpaired Mann–Whitney U test for three biological replicates (mean ± SD). All comparisons are relative to siNT.
Article Snippet: To measure the ratio of mature 28S to 18S rRNAs, total RNA that was prepared as described above was run on an Agilent Technologies 2100 Bioanalyzer at the Yale Center for Genome Analysis.
Techniques: Transfection, Nucleic Acid Electrophoresis, Northern Blot, Labeling, One-tailed Test, MANN-WHITNEY