During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

Fragile X syndrome is caused by the absence of protein FMRP, the function of which is still poorly understood. Previous studies have suggested that FMRP may be involved in various aspects of mRNA metabolism, including transport, stability and/or translatability. FMRP was shown to interact with a subset of brain mRNAs as well as with its own mRNA; however, no speci®c RNA-binding site could be identi®ed precisely. Here, we report the identi®cation and characterization of a speci®c and high af®nity binding site for FMRP in the RGG-coding region of its own mRNA. This site contains a purine quartet motif that is essential for FMRP binding and can be substituted by a heterologous quartet-forming motif. The speci®c binding of FMRP to its target site was con®rmed further in a reticulocyte lysate through its ability to repress translation of a reporter gene harboring the RNA target site in the 5¢-untranslated region. Our data address interesting questions concerning the role of FMRP in the post-transcriptional control of its own gene and possibly other target genes.

The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerizatlon of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides A ubiquitous property of retroviruses is that their genonme consists of two homologous copies of single-stranded RNA (1). Electron microscopy showed that these two RNAs are joined together in an apparent parallel orientation by a structure called the dimer linkage structure (DLS) located near their 5' end (2-5). Dimerization of genomic RNA is considered to control several essential steps of the retroviral life cycle. First, it was proposed to act as a positive signal for encapsidation (6,7). Second, it was suggested to downregulate the translation ofthe gag gene in Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) (8, 9). Third, the dimeric nature of the retroviral genome is thought to be of importance in the process of reverse transcription and recombination since it may account for the first strand transfer and template switching during proviral DNA synthesis (10-14). Therefore, dimerization of genomic RNA most likely represents a potential target for the design of antiviral drugs against HIV and other retroviruses.However, the process of dimerization is still poorly understood. Dimerization of synthetic fragments containing the (29). Again, probing data indicate that the same structure is found in synthetic RNA fragments (9,30) and in genomic RNA extracted from infected cells (29).In HIV-1, several reports showed that a RNA region of -100 nucleotides located downstream of the splice donor (SD) site is able to dimerize in vitro (6,(21)(22)(23)(24). This region that includes important components of the packaging signal (29,31,32) was assumed to contain the putative DLS. However, the mechanism of dimerization is still a subject of controversy. In an initial study, we proposed that polypurine tracts, the only common motifs found in the putative dimerization-encapsidation region of most retroviral RNAs, may be involved in the dimerization process through the formation of quartets involving both guanines and adenines (21). This concept has been disputed by recent reports suggesting that dimers formed with short RNA restricted to the postulated DLS are stabilized by quartets containing only guanines (23,24). However, the di...