Fragile X premutation rCGG repeats cause the nuclear accumulation of Hsp70 mRNA.

Abstract

<p>A. Quantitative analysis of <i>Hsp70</i> mRNA levels by real-time PCR from the adult heads of genotypes: <i>+/+</i> (wild-type (WT)); <i>elav; rCGG₆₀</i> (rCGG-expressing homozygous flies); <i>elav/+; rCGG₆₀/+</i> (rCGG-heterozygous flies); <i>Rm62^LOF/+</i> (Rm62 mutation heterozygous flies); and <i>rCGG₆₀/+; Rm62^LOF/+</i> (interaction). Housekeeping ribosomal protein 32 (<i>Rpl32</i>) mRNA was used as an internal control. *: p<0.05 B. Western blot with anti-histone 3 antibody, and α tubulin. C. Quantitative analysis of <i>Hsp70</i> mRNA levels by real-time PCR on cytoplasmic and nuclear RNA fractions obtained from adult heads of wild-type (WT) and rCGG-expressing flies. <i>Rpl32</i> mRNA was used as control. D. Quantitative analysis of <i>Hsp70</i> mRNA levels in total RNA fractions upon heat shock. Both wild-type (WT) and rCGG-expressing flies were heat shocked for 30 min. No heat shock (NHS) represents non-heat shocked controls. Flies were decapitated at the indicated time after heat shock. Heads were collected and total RNA isolated from them. Both WT and rCGG-expressing flies displayed robust expression of <i>Hsp70</i> in response to heat shock. After the removal of heat shock, <i>Hsp70</i> transcripts declined radically in the WT, whereas in rCGG-expressing flies, <i>Hsp70</i> transcripts show prolonged accumulation. Control samples do not display any overt differences in the timing or expression levels of <i>Hsp70</i> during the initial response to a short heat shock. Real time against <i>Fmr1</i> serves as a control on fractionated samples. The data represent mean ± SEM, n = 3.</p