Epistemic_Statements  

CORD-19:003f719a05b7a35ea4b778ded17cae230f5a0343 JSONTXT 9 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function

Id Subject Object Predicate Lexical cue
T1 185-285 Epistemic_statement denotes Consequently, the mechanisms and factors involved in their replication have been difficult to study.
T2 286-441 Epistemic_statement denotes In an attempt to analyze the cis-and trans-acting factors that could have a role in NV replication, the 3 0 -untranslated region of the genome was studied.
T3 822-1009 Epistemic_statement denotes Since La, PTB, and PABP are important trans-acting factors required for viral translation and replication, these RNA-protein interactions may play a role in NV replication or translation.
T4 1253-1456 Epistemic_statement denotes Noroviruses are difficult to study because they cannot be cultivated in cell culture; however, molecular techniques have been useful to examine the genome organization and function of the viral proteins.
T5 1632-1683 Epistemic_statement denotes The presence of a 5 0 UTR has not been established.
T6 1905-2175 Epistemic_statement denotes The mechanisms and factors involved in NV translation and RNA replication remain unknown; however, as a positive-stranded RNA virus, the genomic RNA has to be translated into the viral non-structural proteins and function as a template for negative-strand RNA synthesis.
T7 2638-2856 Epistemic_statement denotes NV and poliovirus (PV) share a high level of homology between their non-structural proteins, and their polyadenylated tails; thus, it is possible that both RNA viruses use similar mechanisms to replicate their genomes.
T8 2857-3012 Epistemic_statement denotes The poly(A) tail increases infectivity [11] and has been considered an important cis-acting element required for negative-strand RNA synthesis of PV [12] .
T9 3013-3222 Epistemic_statement denotes This element is located at the 3 0 end of the genomic RNA, where the initiation of the RNA synthesis takes place and has a critical role in organizing proteins around the start site of the RNA synthesis [12] .
T10 4141-4341 Epistemic_statement denotes Structural and functional similarities in the replication process among polyadenylated RNA viruses suggest that NV may share some of the elements and follow related strategies to replicate its genome.
T11 4342-4517 Epistemic_statement denotes In order to characterize some elements that could have a role in NV RNA synthesis, the interactions between the 3 0 UTR, the poly(A) tail, and cellular proteins were analyzed.
T12 4518-4696 Epistemic_statement denotes The present results indicate that NV 3 0 UTR with a 24 poly(A) tail (3 0 UTR(A)) is able to form a stable stemloop structure of 44 nts as predicted by the mfold-2 software [22] .
T13 4817-4963 Epistemic_statement denotes La from HeLa cells and a recombinant PTB specifically interact with the NV 3 0 UTR (3 0 UTR) while the PABP exclusively binds to the poly(A) tail.
T14 6672-6739 Epistemic_statement denotes HeLa cells were prepared using a method previously described [24] .
T15 6778-6871 Epistemic_statement denotes Mobility shift electrophoresis assay was performed using a method previously described [23] .
T16 7349-7389 Epistemic_statement denotes UV cross-linking of RNA-protein complex.
T17 7390-7611 Epistemic_statement denotes UV-induced crosslinking assay of RNA-protein complexes was performed using a method previously described [24] in the presence of 40 or 60 lg of S10 extract from HeLa cells and 100 or 500 ng of the recombinant PTB protein.
T18 7681-7796 Epistemic_statement denotes Immunoprecipitation of the cross-linked La-protein complex was performed using a method previously described [23] .
T19 8002-8169 Epistemic_statement denotes The stem-loop structure formed with the last 47 nts has a DG ¼ À9:6, suggesting its stability and was not altered in the presence of the 24 nts long poly(A) tail (Fig.
T20 8176-8399 Epistemic_statement denotes In order to determine if the 3 0 UTR was able to interact with cellular proteins present in HeLa cell extract, mobility shift assays were performed using [a-32 P]UTP labeled RNA representing the complete 3 0 UTR as a probe.
T21 8400-8491 Epistemic_statement denotes Under this condition, a major RNA-protein complex was observed as a detectable smear ( Fig.
T22 8630-8780 Epistemic_statement denotes Furthermore, when the RNA-protein complex was treated with RNase before electrophoresis through the native gel, two main complexes were observed (Fig.
T23 8798-8866 Epistemic_statement denotes The major complex, with the fastest migration, was called complex I.
T24 8867-9006 Epistemic_statement denotes To determine the stability of both RNA-protein complexes, complex formation was performed in the presence of increasing KCl concentrations.
T25 9210-9274 Epistemic_statement denotes These results suggest that both complexes, I and II, are stable.
T26 9376-9606 Epistemic_statement denotes 2D ), using 10-and 20-fold molar excess of unlabeled homologous or a non-related heterologous (referred to as nts 111-191 from NV that do not interact with cellular proteins as has been described before [23] ) RNAs as competitors.
T27 9808-9928 Epistemic_statement denotes However, a 20-fold molar excess of unlabeled non-related heterologous RNA transcript was an inefficient competitor (Fig.
T28 9943-10063 Epistemic_statement denotes These results strongly suggest that the proteins present in HeLa cell S10 extract bound specifically to the 3 0 UTR RNA.
T29 10064-10232 Epistemic_statement denotes To determine whether the poly(A) tail was also involved in the complex formation, an [a-32 P]UTP labeled 3 0 UTR(A) was incubated with S10 extract from HeLa cells (Fig.
T30 10385-10700 Epistemic_statement denotes The complexes formed with both RNAs could be different since the poly(A) tail is a target for the binding of cellular proteins; however, RNP complexes were treated with RNAses, thus neither the poly(A) tail nor the complex formed with it, which were not labeled with [a-32 P]UTP, can be detected by autoradiography.
T31 10803-10965 Epistemic_statement denotes Under these conditions, we could detect differences in the complex (lane 1) or with 5, 10, 15, and 20 lg of S10 extract from HeLa cells (lanes 2-5, respectively).
T32 11663-11774 Epistemic_statement denotes Complex formation was assayed by electrophoresis on native polyacrylamide gels and detected by autoradiography.
T33 12141-12332 Epistemic_statement denotes However, complex II formed with the 3 0 UTR(A), which together with complex V are the most prominent complexes formed with this RNA, did not correspond to any complex formed with the 3 0 UTR.
T34 12516-12769 Epistemic_statement denotes These results suggest that the poly(A) tail may influence the formation of the RNAprotein complexes either because it changes the size and conformation of the RNA and/or because it interacts with some other proteins present in the S10 HeLa cell extract.
T35 12770-12954 Epistemic_statement denotes To determine if the poly(A) tail was able to interact with some HeLa cell proteins, competition experiments using both unlabeled RNAs and a non-related heterologous RNA were performed.
T36 13412-13557 Epistemic_statement denotes Moreover, the amount of radioactivity present in complex II was higher than the amount present in complex II formed without competitor RNAs (Fig.
T37 13594-13695 Epistemic_statement denotes These results suggest that complex II contains proteins, which bind specifically to the poly(A) tail.
T38 13696-13867 Epistemic_statement denotes The addition of a 25-fold molar excess of the unlabeled non-related heterologous RNA was able to compete with complex V, suggesting that this complex is not specific (Fig.
T39 13882-14032 Epistemic_statement denotes Since PABP interacts with the poly(A) tail of several mRNAs, the possibility that complex II contained the PABP was analyzed using a supershift assay.
T40 14266-14385 Epistemic_statement denotes The absence of complex II in the presence of the anti-PABP antibodies demonstrated that PABP was present in complex II.
T41 14709-14799 Epistemic_statement denotes 4C, lane 2) , indicating that the rPABP was able to specifically bind to the poly(A) tail.
T42 14800-14980 Epistemic_statement denotes It has been reported that PABP can bind to the PV 3 0 UTR with a poly(A) tail containing a minimum of eight nts and that its affinity increases as the poly(A) tail lengthens [12] .
T43 14981-15103 Epistemic_statement denotes The poly(A) tail present in the 3 0 UTR(A) is 24 nt long, therefore more than one PABP molecule can bind to this sequence.
T44 15104-15196 Epistemic_statement denotes The two complexes observed after the RNAse could contain different defined amounts of rPABP.
T45 15798-16056 Epistemic_statement denotes It was not possible to detect any differences in the proteins crosslinked to both RNAs; however, a higher amount of radioactivity was observed in the 68 kDa protein crosslinked to the 3 0 UTR(A) (7%), compared to the same band crosslinked to the 3 0 UTR RNA.
T46 16057-16175 Epistemic_statement denotes The increased amount of radioactivity in the 68-kDa band, could correspond to the PABP that binds to the poly(A) tail.
T47 16176-16281 Epistemic_statement denotes To examine this possibility, a 50-fold molar excess of the homologous and the heterologous RNAs was used.
T48 16357-16474 Epistemic_statement denotes 5B, lanes 4 and 3, respectively) ; however, the 68-kDa protein competed only with the homologous unlabeled RNA (Fig.
T49 16490-16702 Epistemic_statement denotes These results suggest that the presence of the poly(A) tail was responsible for the competition of an extra band of 68-kDa, which co-migrates with the 68 kDa band bound to the [a-32 P] ATP labeled 3 0 UTR(A) RNA.
T50 17194-17362 Epistemic_statement denotes To investigate if La protein could be one of the proteins that crosslinked to the 3 0 UTR, an immunoprecipitation after the UV-induced crosslinking assay was performed.
T51 17678-17815 Epistemic_statement denotes To analyze the possible interaction of PTB with the NV 3 0 UTR, labeled 3 0 UTR was incubated with a recombinant PTB (rPTB) protein (Fig.
T52 17919-18068 Epistemic_statement denotes However, this rPTB showed a migration slightly different from that of the 57/60 kDa protein from HeLa cell extracts crosslinked to the same RNA (Fig.
T53 18446-18568 Epistemic_statement denotes These results strongly suggest that the 3 0 UTR contains structural elements that permit the interaction with La and rPTB.
T54 18569-18833 Epistemic_statement denotes Most of the mechanisms and factors involved in NV translation and replication remain unknown; however, as a positive-stranded RNA virus, the viral genome must function as a mRNA for viral protein synthesis and as a template for viral negative-strand RNA synthesis.
T55 19379-19540 Epistemic_statement denotes In the replication process, the presence of cellular proteins could help to recruit and stabilize the RdRp on the initiation sites for viral RNA synthesis [15] .
T56 19946-20110 Epistemic_statement denotes Genome circularization has a critical role in the coordination of translation and RNA synthesis and in the location of the RdRp at the appropriate start site [12] .
T57 20266-20430 Epistemic_statement denotes Using the mfold-2 software, it was possible to predict that the 66 nts present in the complete NV 3 0 UTR contained a stem-loop structure formed in the last 47 nts.
T58 20431-20562 Epistemic_statement denotes It was demonstrated that in the absence of RNAse, the 3 0 UTR was able to form a large RNA-protein complex with HeLa cell proteins.
T59 20832-20987 Epistemic_statement denotes The specificity of the complex formation was analyzed in competition experiments performed in the presence of an excess of homologous or heterologous RNAs.
T60 20988-21270 Epistemic_statement denotes A total reduction of complex II and a significant reduction of complex I formed with the 3 0 UTR and HeLa cell S10 extract were observed when samples were incubated in the presence of homologous, but not heterologous competitor, strongly suggesting that both complexes are specific.
T61 21574-21759 Epistemic_statement denotes We could not detect any difference in the RNP complexes formed with the [a-32 P]UTP 3 0 UTR or 3 0 UTR(A), even though the poly(A) tail is a target for the binding of cellular proteins.
T62 21760-22017 Epistemic_statement denotes This finding could be the result of the RNAse treatment, where the poly(A) tail, which was not labeled with the [a-32 P]UTP, alone or bound to cell proteins, could be separated from the labeled complexes and consequently was not detected by autoradiography.
T63 22139-22370 Epistemic_statement denotes Under this condition, we detected the formation of five complexes with both RNAs, some of them with the same migration patterns, but complex II, formed with the 3 0 UTR(A), did not correspond to any complex formed with the 3 0 UTR.
T64 22371-22470 Epistemic_statement denotes Then complex II could be formed by the poly(A) tail bound to proteins present in HeLa cell extract.
T65 22636-22793 Epistemic_statement denotes We could demonstrate that complex II contains proteins that specifically bind to the poly(A) tail, since this complex was not completed with the 3 0 UTR RNA.
T66 22794-22918 Epistemic_statement denotes Moreover, the presence of the PABP in complex II was confirmed in mobility gel supershift assays using anti-PABP antibodies.
T67 23039-23230 Epistemic_statement denotes The two bands in the mobility shift assay can be explained due to the presence of the 24 nts long tail that could permit the formation of a RNP complex with more than one PABP molecule [12] .
T68 23231-23337 Epistemic_statement denotes Then RNAse treatment could generate two different complexes containing different defined amounts of rPABP.
T69 23453-23567 Epistemic_statement denotes However, no difference between both patterns at the usual electrophoretic migration of PABP (68-72 kDa) was found.
T70 23568-23976 Epistemic_statement denotes Even so, the 68 kDa band that crosslinks with the NV3 0 UTR(A) contains more that one protein, since the competition experiments using an excess of homologous RNA competed completely with this band; while the heterologous RNA, which does not contain the poly(A) tail, only competed partially, suggesting that the 68 kDa band contains at least one protein that specifically binds to the poly(A) tail sequence.
T71 24257-24522 Epistemic_statement denotes This is an interesting finding because both proteins are also associated to some viral functions: La has an essential role in PV translation [29] while PTB is necessary for PV and hepatitis C (HCV) translation [16, 30] and mouse hepatitis virus RNA synthesis [31] .
T72 24523-24975 Epistemic_statement denotes Even though there is no evidence about the mechanisms that operate in NV replication, the work reported here provides new data pointing towards the identification of some of the elements that could have a role in these processes, such as the presence of a stem-loop structure within the polyadenylated 3 0 UTR of NV genomic RNA, and the identification of some of the cellular proteins that specifically bind to this region, including La, PTB, and PABP.
T73 24976-25221 Epistemic_statement denotes The identification of these cis-and trans-elements, considered molecules that have essential roles in translation and replication of other positive-sense RNA viruses, can help understand the specific mechanisms that take place in NV replication.