NXF1 and CRM1 nuclear export pathways orchestrate nuclear export, translation and packaging of murine leukaemia retrovirus unspliced RNA.

Cellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukaemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending on the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.

Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles.

HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5'UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5'UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5'UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles.

Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA.

In Moloney murine leukemia virus, the encapsidation Psi element was shown to be necessary and sufficient to promote packaging of viral RNA, and to be required for dimerization. The conformation of the Psi domain (nucleotides 215 to 565) was investigated in solution by chemical probing. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate at cytosine N3 and adenosine N1, and with a carbodiimide derivative at guanosine N1 and uridine N3. Position N7 of adenine residues was probed with diethylpyrocarbonate. The analyses were conducted on in vitro transcribed fragments corresponding either to the isolated Psi domain or to the 5'-terminal 725 nucleotides. The RNA fragments were analyzed in their monomeric and dimeric forms. A secondary structure model was derived from probing data, computer prediction and sequence analysis of related murine retroviruses. One major result is that Psi forms an independent and highly structured domain. Dimerization induces an extensive reduction of reactivity in region 278 to 309 that can be interpreted as the result of intermolecular interactions and/or intramolecular conformational rearrangements. A second region (around position 215) was shown to display discrete reactivity changes upon dimerization. These two regions represent likely elements of dimerization. More unexpectedly, reactivity changes (essentially enhancement of reactivity) were also detected in another part of Psi (around position 480) not believed to contain elements of dimerization. These reactivity changes could be interpreted as dimerization-induced allosteric transitions.

Target site of Escherichia coli ribosomal protein S15 on its messenger RNA. Conformation and interaction with the protein.

The regulatory site of ribosomal protein S15 has been located in the 5' non-coding region of the messenger, overlapping with the ribosome loading site. The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1. Our results show that the RNA is organized into three domains. Domains I and II, located in the 5' part of the mRNA transcript, are folded into stable stem-loop structures. The 3'-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations. One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired. An alternative potential structure involves a "pseudo-knot" interaction between bases of this domain and bases in the loop of domain II. The conformation of several RNA variants has also been investigated. The deletion of the 5'-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains. The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III. The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control. However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure. Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism. Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation. A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation.

Modified nucleotides of tRNAPro restrict interactions in the binary primer/template complex of M-MuLV.

In all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3'-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAPro complex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNAPro complex. In contrast with other retroviruses, we found that the interaction between tRNAPro and the M-MuLV RNA template is restricted to the standard PBS interaction. In this binary complex, the viral RNA is highly constrained and the rest of tRNAPro is rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAPro lacking the post-transcriptional modifications is substituted for the natural tRNAPro primer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNAPro prevent additional contacts between tRNAPro and the U5 region of M-MuLV RNA.

Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site.

We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.

Crosslinking of ribosomal protein S18 to 16 S RNA in E.coli ribosomal 30 S subunits by the use of a reversible crosslinking agent: trans-diamminedichloroplatinum(II).

We have previously developed [(1987) Biochemistry 26, 5200-5208] the use of trans-diamminedichloroplatinum(II) to induce reversible RNA-protein crosslinks in the ribosomal 30 S subunit. Protein S18 and, to a lesser extent, proteins S13/S14, S11, S4 and S3 could be crosslinked to the 16 S rRNA. The aim of the present work was to identify the crosslinking sites of protein S18. Three sites could be detected: a major one located in region 825-858, and two others located in regions 434-500 and 233-297. This result is discussed in the light of current knowledge of the topographical localization of S18 in the 30 S subunit and of its relation with function.

Spontaneous dimerization of retroviral MoMuLV RNA.

The genome of the Moloney murine leukemia virus (MoMuLV) is composed of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Dimerization sequences are located within the PSI encapsidation domain. We present here an overview of the work we have performed on spontaneous dimerization of a MoMuLV RNA fragment encompassing the PSI domain in order to understand the mechanism by which retroviral RNA dimerization takes place. We present kinetical, thermodynamical and conformational evidence which leads to the conclusion that the PSI domain is a structurally independent domain and that conformational changes are triggered by the dimerization process. We conclude that at least one particular region (nucleotides 278-309) of the RNA is directly involved in the process while the conformation of some other regions is changed probably because of a long-range effect.

Higher-order structure of domain III in Escherichia coli 16S ribosomal RNA, 30S subunit and 70S ribosome.

We have investigated in detail the conformation of domain III of 16S rRNA (nucleotides 913-1408), using a variety of chemical and enzymatic structure probes. The sites of reaction were identified by primer extension with reverse transcriptase using appropriate oligodeoxyribonucleotide primers. This study has been done on 16S rRNA in its naked form, in the 30S subunit and in the 70S ribosome. Data obtained with naked RNA broadly confirm the secondary structure model proposed essentially by comparative sequence analysis, and allow identification of nucleotides involved in tertiary interactions. Our results are in reasonably good agreement with structure probing experiments of Moazed et al. [1]. However, several discrepancies have been observed. Within the 30S subunit, a high number of nucleotides become unreactive whereas other nucleotides show an enhanced reactivity. This probably reflects local conformational changes. Interestingly, they are located in strategic regions of the RNA, e.g. around C1400 (involved in tRNA binding) and C1192 (involved in spectinomycin recognition). Results are also discussed together with the topographical localization of the ribosomal proteins in this area. The study on the 70S particle allows identification of regions at the interface of subunits or exposed at the surface of the ribosome.

A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions.

Four putative hairpin structures (hairpins A to D) are involved in the specific encapsidation of Moloney murine leukemia virus (M-MuLV) RNA into M-MuLV virus particles. The C and D elements, encompassing M-MuLV viral nucleotides 310 to 374, facilitate encapsidation of heterologous RNA into virions. Thus, these two elements appear to act as a core RNA encapsidation signal. The loop sequences of the putative C and D hairpins are identical (GACG). However, when GACG loops were introduced into RNAs on heterologous stem sequences, they increased encapsidation levels only three- to fourfold. These results suggest that C and D stem-and-loop sequences contribute to the M-MuLV cis-acting site for encapsidation.

Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization.

A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA. In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1. The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant. Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint. The association is only slightly affected by temperature (delta H = -1.8 kcal/mol). The entropy change [delta S = +29 cal/(mol K)] is clearly the main driving force for the reaction. The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium. The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8. An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded. On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature. The effect of pH on both temperature and ionic strength dependence of Ka has been examined. It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect. On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding. Furthermore, the unique cysteine present in S8 was shown to be essential for binding.

cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA.

We have analyzed the roles of RNA structural motifs located in the 5' part of the Moloney murine leukemia virus (M-MuLV) encapsidation domain (Psi region) with regard to their effects on viral replication. Four putative stem-loop structures between the 5' splice donor site and the gag initiation codon have been examined: stem structure A, corresponding to M-MuLV viral nucleotides 211 to 224; stem-loop B, nucleotides 278 to 303; stem-loop C, nucleotides 310 to 352; and stem-loop D, nucleotides 355 to 374. By measuring infectivities, encapsidation and splicing efficiencies, and endogenous reverse transcription levels of motif A, B, C, and D deletion mutants, we identified mutations which affect replication at the encapsidation step. In particular, deletion of all four motifs in a single mutant eliminated encapsidation of viral RNA, while deletion of individual elements moderately reduced the encapsidation efficiencies. Through analysis of different deletion combinations, we found that deletion of the first two motifs (A plus B) reduced both encapsidation and reverse transcription efficiencies, while deletion of the 3' motifs (C plus D) eliminated encapsidation. Interestingly, the C and D motifs both contain a GACG loop sequence and are highly conserved among murine type C retroviruses. Our results indicate that M-MuLV motifs C and D are necessary for efficient encapsidation, and the presence of at least one of these two stem-loops is crucial to encapsidation and virus replication.

The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.

A novel subgenomic murine leukemia virus RNA transcript results from alternative splicing.

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD') within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD' was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD' without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.

The ribosomal protein S8 from Thermus thermophilus VK1. Sequencing of the gene, overexpression of the protein in Escherichia coli and interaction with rRNA.

The gene of the ribosomal protein S8 from Thermus thermophilus VK1 has been isolated from a genomic library by hybridization of an oligonucleotide coding for the N-terminal amino acid sequence of the protein, amplified by PCR and sequenced. Nucleotide sequence reveals an open reading frame coding for a protein of 138 amino acid residues (M(r) 15,839). The codon usage shows that 94% of the codons possess G or C in the third position, and agrees with the preferential usage of codons of high G+C content in the bacteria of the genus Thermus. The amino acid sequence of the protein shows 48% identity with the protein from Escherichia coli. Ribosomal protein S8 from T. thermophilus has been expressed in E. coli under the control of the T7 promoter and purified to homogeneity by heat treatment of the extract followed by cation-exchange chromatography. Conditions were defined in which T. thermophilus protein S8 binds specifically an homologous 16S rRNA fragment containing the putative S8 binding site with an apparent association constant of 5 x 10(7) M-1. The overexpressed protein binds the rRNA with the same affinity as that extracted from T. thermophilus, indicating that the thermophilic protein is correctly folded in E. coli. The specificity of this binding is dependent on the ionic strength. The protein S8 from T. thermophilus recognizes the E. coli rRNA binding sites as efficiently as the S8 protein from E. coli. This result agrees with sequence comparisons of the S8 binding site on the small subunit rRNA from E. coli and from T. thermophilus, showing strong similarities in the regions involved in the interaction. It suggests that the structural features responsible for the recognition are conserved in the mesophilic and thermophilic eubacteria, despite structural peculiarities in the thermophilic partners conferring thermostability.

Role of conserved nucleotides in building the 16S rRNA binding site of E. coli ribosomal protein S8.

Ribosomal protein S8 specifically recognizes a helical and irregular region of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a question of debate. Here, we used chemical probing to analyze the structural consequences of mutations in this RNA region. These data, combined with computer modelling and previously published data on protein binding were used to investigate the conformation of the RNA binding site. The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already proposed non canonical U598-U641 interaction, the structure is stabilized by stacking interactions (between A595 and A640) and an array of hydrogen bonds involving bases and the sugar phosphate backbone. Mutations that alter the ability to form these interdependent interactions result in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted backbone and the two bulged adenines 640 and 642 in the deep groove. The third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598-U641 pair should not be directly in contact with the protein.

Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278-303) and new roles for stem-loops H2 (310-352) and H3 (355-374).

Genomic RNAs from retroviruses are packaged as dimers of two identical RNA molecules. In Moloney murine leukemia virus, a stem-loop structure (H1) located in the encapsidation domain Psi (nucleotides 215-564) was postulated to trigger RNA dimerization through base pairing between auto complementary sequences. The Psi domain also contains two other stem-loop structures (H2 and H3) that are essential for RNA packaging. Since it was suspected than H1 is not the only element involved in RNA dimerization, we systematically investigated the dimerization capacity of several subdomains of the first 725 nucleotides of genomic RNA. The efficiency of dimerization of the various RNAs was estimated by measuring their apparent dissociation constants, and the specificity was tested by competition experiments. Our results indicate that the specificity of dimerization of RNA nucleotides 1-725 is driven by motifs H1-H3 in domain Psi. To define the relative contributions of these elements, RNA deletion mutants containing different combinations of H1-H3 were constructed and further analyzed in competition and kinetic experiments. Our results confirm the importance of H1 in triggering dimerization and shed new light on the mechanism of dimerization. H1 is required to provide a stable dimer, probably through the formation of extended intermolecular interactions. However, H1-mediated association is a slow process that is kinetically enhanced by H3, and to a lesser extent by H2. We suggest that they facilitate the recognition between the two RNAs, most likely through their conserved GACG loops. Our results reinforce the idea that dimerization and packaging are two closely related processes.

Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization.

Dimerization of genomic RNA is a key step in the retroviral life cycle and has been postulated to be involved in the regulation of translation, encapsidation and reverse transcription. Here, we have derived a secondary structure model of nucleotides upstream from psi and of the gag initiation region of Mo-MuLV RNA in monomeric and dimeric forms, using chemical probing, sequence comparison and computer prediction. The 5' domain is extensively base-paired and interactions take place between U5 and 5' leader sequences. The U5-PBS subdomain can fold in two mutually exclusive conformations: a very stable and extended helical structure (E form) in which 17 of the 18 nucleotides of the PBS are paired, or an irregular three-branch structure (B form) in which 10 nucleotides of the PBS are paired. The dimeric RNA adopts the B conformation. The monomeric RNA can switch from the E to the B conformation by a thermal treatment. If the E to B transition is associated to dimerization, it may facilitate annealing of the primer tRNAPro to the PBS by lowering the free energy required for melting the PBS. Furthermore, dimerization induces allosteric rearrangements around the SD site and the gag initiation region.

Minimal 16S rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition.

Escherichia coli ribosomal protein S8 was previously shown to bind a 16S rRNA fragment (nucleotides 584-756) with the same affinity as the complete 16S rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezançon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91-107]. Region C was postulated to display characteristic features: three bulged adenines (A595, A640 and A642), a non-canonical U598-U641 pair surrounded by two G.C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588-605/633-651) containing region C. The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site-directed mutagenesis in fragment 584-756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597.C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.

Probing the structure of RNAs in solution.

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.