Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles

Abstract

Fragile X syndrome is caused by the absence of the fragile X mental retardation protein (FMRP). This RNA-binding protein is widely expressed in human and mouse tissues, and it is particularly abundant in the brain because of its high expression in neurons, where it localizes in the cell body and in granules throughout dendrites. Although FMRP is thought to regulate trafficking of repressed mRNA complexes and to influence local protein synthesis in synapses, it is not known whether it has additional functions in the control of translation in the cell body. Here, we have used recently developed approaches to investigate whether FMRP is associated with the translation apparatus. We demonstrate that, in the brain, FMRP is present in actively translating polyribosomes, and we show that this association is acutely sensitive to the type of detergent required to release polyribosomes from membranous structures. In addition, proteomic analyses of purified brain polyribosomes reveal the presence of several RNA-binding proteins that, similarly to FMRP, have been previously localized in neuronal granules. Our findings highlight the complex roles of FMRP both in actively translating polyribosomes and in repressed trafficking ribonucleoparticle granules.

Fig. 1.

Analyses of brain polyribosomes prepared from adult mice. (a) Postmitochondrial fraction was treated with 1% nonionic detergent Nonidet P-40, and total polyribosomes were first concentrated by ultracentrifugation, resuspended, and analyzed by sedimentation velocity throughout sucrose density gradients. Liver polyribosomes treated in the same way were analyzed in parallel. Note the presence of brain polyribosomes and FMRP at the bottom of the centrifuge tube, whereas the distribution of liver polyribosomes is different. (b) Treatment with 1% DOC allows brain and liver polyribosomes to distribute throughout the gradients according to their sedimentation values, whereas FMRP is detected at the top of the gradients. The integrity and distribution of polyribosomes were based on the UV profile as well as the presence of L7, a core protein of the large ribosomal subunit. Distribution of FMRP in different fractions was revealed by immunoblotting with mAb1C3.

Fig. 2.

Effects of Nonidet P-40 (Left) and DOC (Right) on brain polyribosomes prepared from young animals. Although no major differences can be detected between the two polyribosomal profiles, the distribution of FMRP is clearly affected after treatment with DOC.

Fig. 3.

Schematic diagram of the steps used to prepare polyribosomes from the postmitochondrial supernatant and the residual fractions derived from homogenates of young mouse brain.

Fig. 4.

Quantitative distribution of brain polyribosomes and FMRP from the fractions prepared by differential sedimentations, as described in Fig. 3. Note that most of FMRP is associated with heavy sedimenting polyribosomes (P1), whereas only trace amounts are detected at the top of the gradient in panel FP, which stands for the final pellet obtained after centrifugation of the postribosomal pellet for 18 h at 105,000 × g.

Fig. 5.

FMRP and several other RNA-binding proteins are released from polyribosomes after treatment with DOC, whereas Nonidet P-40 has no deleterious effects on the purification of polyribosomes that still carry these RNA-binding proteins. Immunoblot analyses were performed with the indicated specific antibodies. Note that neither Nonidet P-40 nor DOC has an effect on the core ribosomal proteins S6 and L7. P, polyribosomes recovered after ultracentrifugation; S, protein soluble either in Nonidet P-40 or DOC.

Fig. 6.

Identification of proteins extracted from polyribosomes after treatment with 1% DOC. The major Coomassie brilliant blue-stained bands in lane S were eluted and analyzed by MS. Identified proteins are classified as RNA-binding proteins and other proteins. P, polyribosomal proteins resistant to DOC treatment; S, soluble proteins after DOC treatment.