Cytoplasmic RNA-binding proteins and the control of complex brain function

Abstract

The formation and maintenance of neural circuits in the mammal central nervous system (CNS) require the coordinated expression of genes not just at the transcriptional level, but at the translational level as well. Recent evidence shows that regulated messenger RNA (mRNA) translation is necessary for certain forms of synaptic plasticity, the cellular basis of learning and memory. In addition, regulated translation helps guide axonal growth cones to their targets on other neurons or at the neuromuscular junction. Several neurologic syndromes have been correlated with and indeed may be caused by aberrant translation; one important example is the fragile X mental retardation syndrome. Although translation in the CNS is regulated by multiple mechanisms and factors, we focus this review on regulatory mRNA-binding proteins with particular emphasis on fragile X mental retardation protein (FMRP) and cytoplasmic polyadenylation element binding (CPEB) because they have been shown to be at the nexus of translational control and brain function in health and disease.

Figure 1.

Basic scheme of the HITS-CLIP method. Beginning at the upper left and moving counterclockwise, tissue (e.g., brain) or cells is UV irradiated at 254 nm introducing covalent cross-links between RNA-protein complexes in living cells. 1. Cell lysis and partial RNase digestion reduces the modal size of cross-linked RNA “tags” to a size determined by the experimenter; 50–100 nucleotides is ideal. RNase A and T1 leave a 5′-OH and a 3′ phosphate group on the digested RNA. 2. Cross-linking allows stringent immunoprecipitation of RNA-binding protein:RNA complexes. 3 and 4. The 3′ phosphate is removed by alkaline phosphatase, to prevent intramolecular RNA circularization during the ligation of a linker to the 3′ end of the RNA tags in step 4. This linker is blocked at the 3′ end with a puromycin molecule to prevent competing linker-linker ligation reactions. 5. The RNA tags are then labeled at the 5′ end with T4 polynucleotide kinase and ³²P-γ-ATP. 6. RNABP:RNA complexes are released from beads, run on denaturing SDS-PAGE, transferred to nitrocellulose, and imaged by autoradiography. SDS-PAGE and transfer to nitrocellulose are two important additional purification steps to separate the desired RNABP:RNA complexes away from other RNABPs or free RNA. 7. The radioactive RNABP:RNA complex is excised from the nitrocellulose filter, digested with proteinase K to remove the RNABP and to elute the RNA, which is then isolated by phenol-chloroform extraction and ethanol precipitation. 8. A second linker is added to the 5′ end of the RNA. 9. The RNA is amplified by RT-PCR and sequenced by high-throughput sequencing methods. The sequences of the tags can then be aligned with the genome of interest to produce a genome-wide map of where the RNABP was bound to RNA in vivo.

Figure 2.

FMRP stalls ribosomes during elongation to repress protein synthesis. (Upper panel) Active translation: In the absence of FMRP, brain transcripts are shown being translated into protein by translocating ribosomes (made up of 40S and 60S subunits shown in light blue), which assemble at the start codon (i.e., AUG, initiation) and dissociate at the stop codon (i.e., UAG, termination). Ribosomal protein P0 is shown as a darker blue sphere on the 60S subunits. The poly(A)-binding protein (PABP) and the Hu family of RNABPs interacting with specific binding sites in 3′ UTRs are depicted with orange and green spheres, respectively. All four of these RNA-binding proteins are polyribosome associated, each by a different mechanism, and for this reason PABP, Hu, and P0 are shown as contrast for the properties and function of FMRP. (Lower panel) Repressed translation is associated with FMRP interaction with target mRNAs. FMRP preferentially interacts with specific mRNAs and in this context inhibits protein synthesis by stalling ribosomal translocation on those transcripts as part of a micrococcal nuclease-resistant multiribosome complex. This inhibition appears to be reversible, as it can be acutely relieved by competing FMRP off of polyribosomes with an RNA decoy; it is unknown whether this might occur in vivo owing to changes in the phosphorylation state of FMRP, its degradation by the proteasome, interactions with other proteins, noncoding RNAs, or other physiologic effectors. The stoichiometry of FMRP and stalled ribosomes remains to be determined. We have drawn a minimum of one (red) FMRP present in the stalled complex, recognizing the possibility that additional FMRP molecules (illustrated by transparent red figures) may be present. The presence of some FMRP in the UTRs (depicted on the 3′ UTR) is consistent with FMRP HITS-CLIP results. (From Darnell et al. 2011; reprinted with modifications, with permission, from Elsevier © 2011.)