Targeting Ribosome assembly on the HCV RNA using a small RNA molecule
Prasanna BhatSivakumar Vadivel GnanasundramPrashant ManiPartho Sarothi RayDebi P. SarkarSaumitra Das
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Abstract:
Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.Keywords:
5.8S ribosomal RNA
The structural complexity of ribosomes is evident from the large number of their constituents - ribosomal proteins and ribonucleic acids. The simplest known prokaryotic (70S) ribosome from E. coli is composed of 53 different proteins and three ribonucleic acid molecules (23S, 16S and 5S RNA). Eukaryotic (80S) ribosomes also consist of three RNAs (28S, 18S and 5S) but contain a higher number of proteins, approximately 70. The mutual interactions of ribosomal proteins and RNAs determine the final structure and thus the biological activity of the ribosome. As expected for particles with the above composition, ribosomes and both ribosomal subunits have no symmetry or repetition in structure. Electron micrographs of ribosomes (stained with 0.5% aqueous uranyl acetate and deposited on a fine carbon support) show distinct contours and electron dense pattern. Critical evaluation of different views of the two-dimensional projections of the ribosomal contours can provide a reasonable basis for proposing a three-dimensional model of the ribosome.
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28S ribosomal RNA
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Canonical cap-dependent translation initiation requires a large number of protein factors that act in a stepwise assembly process. In contrast, internal ribosomal entry sites (IRESs) are cis-acting RNAs that in some cases completely supplant these factors by recruiting and activating the ribosome using a single structured RNA. Here we present the crystal structures of the ribosome-binding domain from a Dicistroviridae intergenic region IRES at 3.1 angstrom resolution, providing a view of the prefolded architecture of an all-RNA translation initiation apparatus. Docking of the structure into cryo-electron microscopy reconstructions of an IRES-ribosome complex suggests a model for ribosome manipulation by a dynamic IRES RNA.
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Amino acid deprivation of a “relaxed” auxotroph of Escherichia coli results in the accumulation of protein-deficient, immature ribosomes (“relaxed particles”). The ribonucleic acid (RNA) of these particles was shown to differ from mature ribosomal RNA in both sedimentation characteristics and in elution from columns of methylated albumin-keiselguhr. When relaxed particles were allowed to become converted to mature ribosomes, the unique properties of the RNA were lost, and this RNA became indistinguishable from mature RNA. The conversion of relaxed particles to ribosomes did not involve degradation and resynthesis of RNA. It is concluded that ribosomal RNA undergoes a configurational transition during ribosome development, and that this transition is not the result of changes in the primary structure of the RNA.
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Abstract Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits in vitro , and presence of the IRES enhanced mRNA association to the ribosomal subunits in whole cell lysates. The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.
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