Results: 2

1.
Figure 1

Figure 1. From: Ribosomal Protein L5 and L11 Mutations Are Associated with Cleft Palate and Abnormal Thumbs in Diamond-Blackfan Anemia Patients.

Pre-rRNA Processing in Human Cells, According to Hadjiolova 49 and Choesmel 22
The mature 18S, 5.8S, and 28S rRNAs are separated by ITS1 and ITS2 and flanked by external transcribed spacers, 5′-ETS and 3′-ETS. The red arrows and the numbers in red indicate the cleavage sites. “A” and “B” represent two pathways of the rRNA processing.

Hanna T. Gazda, et al. Am J Hum Genet. 2008 December 12;83(6):769-780.
2.
Figure 2

Figure 2. From: Ribosomal Protein L5 and L11 Mutations Are Associated with Cleft Palate and Abnormal Thumbs in Diamond-Blackfan Anemia Patients.

Impact of DBA Mutations in RPL5, RPL11, and RPS7 on Pre-rRNA Processing
(A) Northern blot analysis of total RNAs extracted from lymphoblastoid cells with mutations in RPL5 and RPL11. Precursor and mature rRNAs were detected with probes complementary to the ITS2, 18S rRNA, and 28S rRNA sequences. Bar graphs show signal quantification with a phosphoimager and include experiments from additional samples processed separately. Bars indicate variation of 32S/28S and 12S/28S intensity ratio relative to controls (means ± SD). Significance of the difference compared to controls was assessed with a Student's t test assuming nonequal variance (single asterisk indicates p ≤ 0.05; double asterisk indicates p ≤ 0.01; triple asterisk indicates p ≤ 0.001).
(B) Northern blot analysis of total RNAs fractionated on denaturing 6% polyacrylamide gel. After transfer, the blot was successively hybridized with probes complementary either to the junction between 5.8S rRNA and ITS2 (probe 5′-ITS2) or to 5.8S rRNA. 7SK RNA, a stable and abundant noncoding RNA involved the regulation of RNA polymerase II, was chosen as a loading control.
(C) Analysis on sucrose gradient of cytoplasmic ribosomes isolated from HeLa cells 48 hr after transfection with siRNAs directed against the mRNAs encoding RPL5 or RPL11. Arrowheads indicate polysomes containing half-mers. Control: transfection without siRNA. Expression of both siRNAs results in significant reduction of 60S particles. Differences in the extent of reduction are related to differing efficiencies of the siRNAs used.
(D) Northern blot analysis of total RNAs from cells treated as in (C) with probes complementary to the ITS2, the 18S rRNA and the 28S rRNA (1% agarose gel), or with probes complementary to the junction between the 5.8S rRNA and the ITS2 (probe 5′-ITS2) and to the 5.8S (6% polyacrylamide gel). The 7SK RNA is shown as a loading control for the polyacrylamide gel. Scrbl: scramble siRNA.
(E) Northern blot analysis of pre-rRNA processing in lymphoblastoid cell lines (LCL) with mutation in RPS7 and in HeLa cells transfected with siRNAs targeting RPS7 mRNAs. The control RPS7+/+ lymphoblastoid cells are derived from an unaffected sibling of the RPS7+/mut patient. Ctrl: HeLa cells transfected without siRNAs. Methods: The 21 nt siRNA duplexes with a 3′ dTdT overhang, corresponding to L5 mRNA (5′-AAGGGAGCTGTGGATGGAGGC-3′) 47 and to L11 mRNA (5′-AAGGTGCGGGAGTATGAGTTA-3′), 48 were purchased from Eurogentec (Seraing, Belgium) and transfected via electrotransformation, as described previously. 42 Detection of pre-rRNA on northern blots was performed as described previously 50 with oligonucleotidic probes 18S, 28S, ITS1, ITS2b, and ITS2-d/e. 42 For detection of ITS2, ITS2-b and ITS2-d/e probes were mixed in equal amounts. The remaining probes had the following sequences: 5′-ITS1, 5′-CCTCGCCCTCCGGGCTCCGTTAATGATC-3′, 5.8S, 5′-CAATGTGTCCTGCAATTCAC-3′; 5′-ITS2, 5′-GGGGCGATTGATCGGCAAGCGACGCTC-3′, 7SK (mix of two probes), 5′-CATGGAGCGGTGAGGGAGGA-3′, and 5′-GTGTCTGGAGTCTTGGAAGC-3′. For ribosome analysis on sucrose gradient, HeLa cells transfected with siRNAs for 48 hr were treated with 100 mg/ml cycloheximide (Sigma Aldrich, St. Louis, MO) for 10 min and fractionated, and the cytoplasmic fraction was analyzed on a 10%–50% sucrose gradient as described previously. 42

Hanna T. Gazda, et al. Am J Hum Genet. 2008 December 12;83(6):769-780.

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