Approach to identifying the functionally important segments of RNA, based on complementation-addressed modification

An approach based on complementation-addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5'-P and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect, whereas alteration of hairpin IIIe substantially reduced the binding. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play a critical role in the binding of HCV IRES with the 40S subunit.     

The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with pUUUGUU

Three mRNA analogs--derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position--were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA(Phe) was used, while tRNA(Val) was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions -3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with soft UV light (lambda > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions -1 to +3 binds to G1207, while that in positions -2 or -3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the -3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the -3 position, this crosslinking was not observed in the absence of tRNA.     

The mRNA Codon Environment at the P and E Sites of Human Ribosomes Deduced from Photocrosslinking with pUUUGUU Derivatives

Three mRNA analogs—derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position—were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA^Phe was used, while tRNA^Val was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions –3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with mild UV light ( > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions –1 to +3 binds to G1207, while that in positions –2 or –3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the –3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the –3 position, this crosslinking was not observed in the absence of tRNA.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES element) of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group at nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the subunit between the head and the body aside the “beak” near the exit from the mRNA-binding channel.     

The Template Region 5′ of the E-Site Codon Is Close to S26 in the Human 80S Ribosome

The environment of the template sequence 5 of the E-site codon on the 80S ribosome was studied with nonaribonucleotide or dodecaribonucleotide derivatives containing Phe codon UUU at the 3 end and a perfluoroarylazido group at the first or third nucleotide. A photoreactive group was linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which is cognate to the UUU codon and directs it to the P site, bringing a modified nucleotide in position –4 to –9 relative to the first nucleotide of the P-site codon. Upon irradiation of ribosome complexes with tRNA^Phe and the mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins. The major target of modification was S26 in all cases. In addition, S3 was modified to a low extent when the reactive nucleotide was in position –4 and S14 was in position –6. In the absence of tRNA, all mRNA analogs modified S3.     

Part of the matrix on the 5' side from codon in the E-segment is close to protein S26 on the human 80S ribosome

Positioning of mRNA on the 80S ribosome upstream the E site bound codon was studied using derivatives of nona- and dodecaribonucleotides containing the triplet UUU coding for Phe at the 3'-terminus, and a perfluorophenylazide cross-linker on either the first or the third nucleotide. Two sets of the mRNA analogues were used, with the photoactivatable groups on either the C5 atom of the uridine or the N7 atom of the guanosine. The modified nucleotides were directed to positions from -4 to -9 with respect to the first nucleotide of the P site bound codon by tRNA(Phe) cognate to the triplet UUU targeted to the P site. Mild UV-irradiation of ribosomecomplexes with tRNA(Phe) and mRNA analogues resulted in the cross-linking to the 40S subunits preferentially, mainly to the proteins. The principal target for the cross-linking was protein S26 in all cases. Location of the photoactivatable group on the nucleotide at position -4 lead also to the minor cross-linking to protein S3, and at position -6 to protein S14. In the absence of tRNA, all mRNA analogues cross-linked to protein S3.     

Variable and conserved elements of human ribosomes surrounding the mRNA at the decoding and upstream sites

This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modi-fied nucleotides were directed to positions from −9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823–A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.     

Proteins surrounding hairpin IIIe of the hepatitis C virus internal ribosome entry site on the human 40S ribosomal subunit

Binding of the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA to the eIF-free 40S ribosomal subunit is the first step of initiation of translation of the viral RNA. Hairpins IIId and IIIe comprising 253–302 nt of the IRES are known to be essential for binding to the 40S subunit. Here we have examined the molecular environment of the HCV IRES in its binary complex with the human 40S ribosomal subunit. For this purpose, two RNA derivatives were used that bore a photoactivatable perfluorophenyl azide cross-linker. In one derivative the cross-linker was at the nucleotide A296 in hairpin IIIe, and in the other at G87 in domain II. Site-specific introduction of the cross-linker was performed using alkylating derivatives of oligodeoxyribonucleotides complementary to the target RNA sequences. No cross-links with the rRNA were detected with either RNA derivative. The RNA with the photoactivatable group at A296 cross-linked to proteins identified as S5 and S16 (major) and p40 and S3a (minor), while no cross-links with proteins were detected with RNA modified at G87. The results obtained indicate that hairpin IIIe is located on the solvent side of the 40S subunit head on a site opposite the beak.     

An Approach to Identifying the Functionally Important RNA Sites by Complementary Addressed Modification

An approach based on complementary addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5-phosphate and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect on the binding, whereas that of hairpin IIIe substantially reduced it. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play the critical role in the binding of HCV IRES with the 40S subunit.