Epistemic_Statements  

CORD-19:acb5f1a150a07ca259285e3558c2c9a1869fd6af JSONTXT 9 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T1 0-138 Epistemic_statement denotes Characterization of leader-related small RNAs in coronavirus-infected cells: Further evidence for leader-primed mechanism of transcription
T2 149-340 Epistemic_statement denotes Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA.
T3 922-1035 Epistemic_statement denotes They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA.
T4 1036-1203 Epistemic_statement denotes These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template.
T5 1204-1419 Epistemic_statement denotes We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs.
T6 1420-1581 Epistemic_statement denotes These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.
T7 2316-2461 Epistemic_statement denotes Murine coronaviruses are normally associated with hepatitis in vivo and infect a variety of established cell lines in vitro (Wege et al., 1982) .
T8 2919-3032 Epistemic_statement denotes These enzymatic activities are associated with cellular membranes (Brayton et al., 1982 (Brayton et al., , 1984 .
T9 3449-3699 Epistemic_statement denotes In addition to this unique genetic arrangement, it has recently been demonstrated that each mRNA and genomic RNA contain an identical stretch of approximately 72 nucleotides at their 5'-ends (Lai et al., 1983 (Lai et al., , 1984 Spaan et al., 1983) .
T10 3700-3970 Epistemic_statement denotes The presence of a leader RNA common to all viral mRNAs indicates that they are synthesized by joining two noncontiguous RNA segments, since the leader sequences are encoded only at the 5'-end of the genomic RNA (Lai et al., 1983 (Lai et al., , 1984 Spaan et al., 1983) .
T11 3971-4059 Epistemic_statement denotes These data suggest that a unique mechanism of RNA synthesis occurs during MHV infection.
T12 4060-4269 Epistemic_statement denotes This mechanism differs from conventional eukaryotic RNA splicing since UV transcriptional mapping studies indicate that these mRNAs are not derived from cleavage of large precursor RNAs (Jacobs et al., 1981) .
T13 4270-4447 Epistemic_statement denotes The study of replicative-intermediate RNA further suggests that the leader RNA is joined to the mRNAs during transcription, but not post-transcriptionally (Baric et al., 1983) .
T14 4448-4743 Epistemic_statement denotes We have also shown that the double-stranded replicative form (RF) is of genome length, suggesting that the joining mechanism does not involve 'looping out' of intervening sequences in the (-)-stranded RNA template since such a mechanism would have generated subgenomic RFs (Baric et al., 1983) .
T15 5313-5558 Epistemic_statement denotes Presently, the exact length of the leader RNA is not known but must consist of 57 nucleotides from the S-end of the genome plus at most 15 nucleotides which are complementary to the splice junctions on the (-)-stranded RNA (Spaan et al., 1983) .
T16 5852-6018 Epistemic_statement denotes These data support the 'leader-primed' mechanism for coronavirus transcription (Baric et al., 1983) and demonstrate another mode of RNA synthesis in eukaryotic cells.
T17 6019-6072 Epistemic_statement denotes The implications of these findings will be discussed.
T18 11052-11152 Epistemic_statement denotes The sequences underlined represent the common sequences and the possible leader-body junction sites.
T19 11153-11297 Epistemic_statement denotes It has previously been determined that the length of the leader RNA is approximately 72 nucleotides (Armstrong et al., 1984; Lai et al., 1984) .
T20 11778-11964 Epistemic_statement denotes f) , it has been suggested that the splice junction lies within a stretch of 15 nucleotides (AAUCUAAUCUAAACU) (nucleotides 58-72 from the 5'-end) (Lai et al., 1984; Spaan et al., 1983) .
T21 11965-12068 Epistemic_statement denotes However, the exact length of the leader-coding sequences at the 5'-end of the genomic RNA is not clear.
T22 12485-12708 Epistemic_statement denotes The minor DNAs seen in Lane A were probably due to random degradation, or the presence of contaminating truncated leader cDNAs, since longer exposure revealed similar patterns in the undigested leader cDNA control (Lane B).
T23 12709-12773 Epistemic_statement denotes However, minor heterogeneity of genomic RNA cannot be ruled out.
T24 12882-12978 Epistemic_statement denotes These data indicate that a minimum of 72 bases encode the leader RNA at the S-end of the genome.
T25 12979-13036 Epistemic_statement denotes However, the exact length of the leader RNA is not known.
T26 13037-13178 Epistemic_statement denotes We had previously proposed a model of coronavirus mRNA transcription which involved the synthesis of a free leader RNA (Baric et al., 1983) .
T27 13821-13871 Epistemic_statement denotes during infection should also be of similar length.
T28 13872-14048 Epistemic_statement denotes Initial attempts to identify this hypothetical RNA species, labeled in vivo, were unsuccessful, probably due to high backgrounds of tRNA and other small RNAs in infected cells.
T29 14049-14207 Epistemic_statement denotes Furthermore, the free leader RNA was probably present in very small quantities since it would be expected to be constantly sequestered to prime transcription.
T30 14934-15104 Epistemic_statement denotes This result is consistent with the model that the leader RNA is synthesized as a separate entity which is discontinuous from the synthesis of the body sequences of mRNAs.
T31 15727-15810 Epistemic_statement denotes 3C) , these data suggest that they were not derived from degradation of viral RNAs.
T32 15811-16057 Epistemic_statement denotes The presence of these RNAs, which are larger than the 70-82-nucleotide leader RNA, lends further support to a model of mRNA synthesis in which the leader RNA is utilized as a primer, rather than added post-transcriptionally (Baric et al., 1983) .
T33 16200-16399 Epistemic_statement denotes It is not clear, however, whether the leader RNA is released from the (-)-strand RNA template into a cytoplasmic pool, or remains tightly complexed with the RNA polymerase and/or (-)-strand template.
T34 16567-16728 Epistemic_statement denotes It has previously been shown that RNA synthesis takes place in membrane-associated complexes during MHV infection (Brayton et al., 1982 (Brayton et al., , 1984 .
T35 16729-16890 Epistemic_statement denotes Therefore, distribution of the leader RNA in different subcellular compartments should elucidate the mechanism of transcription involving the leader RNA species.
T36 17547-17755 Epistemic_statement denotes This result is consistent with the interpretation that part of the 70-82-nucleotide leader RNA species is coupled to the membrane-associated transcriptional complex and involved in the synthesis of each mRNA.
T37 17756-18048 Epistemic_statement denotes Moreover, although we can not completely rule out the possibility that our fractionation procedures might have disrupted the replication complexes, the detection of a significant portion of these RNA species in cytosol suggests that the leader RNA might also be present in a cytoplasmic pool.
T38 18270-18350 Epistemic_statement denotes 4 ) appear to contain less leader RNA species than those grown at 37°C (Lane 5).
T39 18471-18577 Epistemic_statement denotes Therefore, the synthesis of leader RNA and that of mRNAs could not be uncoupled at these two temperatures.
T40 18578-18836 Epistemic_statement denotes To determine whether the leader-containing RNAs are associated with the RNA template, intracellular RNA from infected cells was extracted and separated by sucrose gradient sedimentation to determine the size of RNA fractions in which leader RNA was detected.
T41 19015-19181 Epistemic_statement denotes 5) , these results suggest that at least some leader RNA species are dissociated from the RNA replicative intermediate, which is 12-328 in size (Baric et al., 1983) .
T42 19182-19349 Epistemic_statement denotes However, this conclusion has to be qualified since The cells were disrupted and separated into nuclear, cytoplasmic (S-100) or membrane (P-100) fractions as described.
T43 19852-20002 Epistemic_statement denotes we can not rule out the possibility that the leader RNA might have been dissociated from the replicative intermediate during the extraction procedure.
T44 20003-20201 Epistemic_statement denotes These data are consistent with the subcellular fractionation studies which suggest that the leader-related RNAs are synthesized as a free entity and may function in viral transcription on membranes.
T45 20202-20396 Epistemic_statement denotes The detection of free leader-containing RNAs in MHV-infected cells provides support for a mechanism of coronavirus transcription which utilizes a small leader RNA as a primer for mRNA synthesis.
T46 20397-20555 Epistemic_statement denotes However, definitive proof of a distinct leader-specific RNA is complicated by the presence of multiple small leader-containing RNAs in infected cell extracts.
T47 20556-20754 Epistemic_statement denotes To provide additional evidence for leader-primed trans~~ption, we reasoned that the synthesis of leader RNA and mRNAs should be discontinuous and, therefore, might require different viral functions.
T48 20755-20853 Epistemic_statement denotes Thus, it might and sedimented at 25 000 rpm on lo-25% sucrose gradients for 16 h in an SW41 rotor.
T49 21204-21311 Epistemic_statement denotes be possible to isolate a temperature-sensitive (ts) mutant which synthesizes only leader RNA but not mRNAs.
T50 22075-22213 Epistemic_statement denotes This is consistent with the observation that these mutants fail to synthesize mRNA at nonpermissive temperature (unpublished observation).
T51 22214-22331 Epistemic_statement denotes However, one mutant, LAlO, synthesizes a series of small leader-related RNA species at the nonpermissive temperature.
T52 23134-23276 Epistemic_statement denotes lated RNAs, these data indicate that the replication of this mutant is blocked at a step after the synthesis of this 72-nucleotide leader RNA.
T53 23277-23442 Epistemic_statement denotes They also suggest that this RNA species is not a premature termination product of mRNA synthesis, and that its synthesis is discontinuous from the synthesis of mRNA.
T54 23923-23995 Epistemic_statement denotes These results are consistent with the RNA-( +) phenotype of this mutant.
T55 23996-24108 Epistemic_statement denotes These results strongly suggest that the synthesis of the leader RNA and mRNAs requires separate viral functions.
T56 24109-24342 Epistemic_statement denotes The presence of a mutant which accumulates leader-specific RNA at nonpermissive temperature lends further support to the model that the leader RNA is synthesized independently and then utilized as a primer for the synthesis of mRNAs.
T57 24343-24577 Epistemic_statement denotes Previous studies on MHV mRNA structure indicate that each viral niRNA is constructed by the joining of two noncontiguous RNA segments encoded on the genome (Baric et al., 1983; Lai et al., 1983 Lai et al., , 1984 Spaan et al., 1983) .
T58 24578-24880 Epistemic_statement denotes Since MHV mRNA synthesis occurs in the cytoplasm and does not involve synthesis and subsequent cleavage of precursor RNAs, viral transcription must occur by a novel mechanism that differs from conventional splicing modes (Baric et al., 1983; Lai et al., 1983; Jacobs et al., 1981; Spaan et al., 1983) .
T59 24881-25099 Epistemic_statement denotes Analysis of MHV replicative intermediate and replicative form RNAs supports this contention and suggests that a free leader RNA is transcribed independently, and then used to prime mRNA synthesis (Baric et al., 1983) .
T60 25323-25583 Epistemic_statement denotes These findings provide strong evidence that MHV leader RNA synthesis is separate from the transcription of the mRNAs and provide additional support for the leader-primed mechanism of coronavirus transcription which we proposed previously (Baric et al., 1983) .
T61 25584-25821 Epistemic_statement denotes Sequence analysis of mRNAs 7 and 6 suggests that the junction sites between the leader RNA and the body sequences of each mRNA lie between nucleotides 58 and 72 from the 5'-end of the genome (Armstrong et al., 1984; Spaan et al., 1983) .
T62 25822-25979 Epistemic_statement denotes From S, mapping studies, the length of the leader-coding sequences at the 5'-end of the genome is at least 72 nucleotides, but the exact length is not known.
T63 26094-26251 Epistemic_statement denotes However, these data were complicated by the presence of several additional discrete leader-specific RNAs which range between 70 and 82 nucleotides in length.
T64 26252-26323 Epistemic_statement denotes Several explanations could account for the multiple leaderrelated RNAs.
T65 26324-26510 Epistemic_statement denotes Analysis of the primary sequence of the leader RNA suggests that several double-stranded RNA loops, similar to tRNA structure, may be present in the leader RNA (unpublished observation).
T66 26511-26647 Epistemic_statement denotes These loops could represent strong stops for RNA synthesis, causing the accumulation of discrete premature termination products in vivo.
T67 26648-26753 Epistemic_statement denotes Alternatively, the different leader RNAs might be primers for transcription of the different viral mRNAs.
T68 26754-26984 Epistemic_statement denotes It has previously been suggested that differences in the degree of complementarity between the 3'-end of leader RNA and different junction sequences on the (-)-strand could regulate the rate of synthesis of each mRNA species (Fig.
T69 27031-27178 Epistemic_statement denotes The requirement for different leader RNAs might also provide an additional mechanism for the regulation of transcription of different mRNA species.
T70 27179-27427 Epistemic_statement denotes The leader-primed transcription model is further supported by our recent finding that the leader sequences could be exchanged at very high frequency between two different MHV strains during mixed infection (Makino et al., unpublished observation) .
T71 27529-27632 Epistemic_statement denotes However, definitive proof that these leader RNAs act as primers awaits an in vitro transcription assay.
T72 27633-27800 Epistemic_statement denotes The larger leader-related RNA species which are longer than 130 nucleotides might represent intermediate RNA products caused by premature termination of transcription.
T73 27801-28012 Epistemic_statement denotes The fact that these RNA species contain the leader sequences and are significantly larger than the 75nucleotide leader RNA provides further evidence that the leader RNA is utilized as a primer for transcription.
T74 28136-28212 Epistemic_statement denotes they can bind back to the RNA template and act as primers for transcription.
T75 28213-28379 Epistemic_statement denotes This possibility has recently been supported by the isolation of recombinant RNA molecules during mixed infection of different MHV strains (unpublished observations).
T76 28553-28676 Epistemic_statement denotes It is possible that two different polymerases are responsible for the synthesis of the leader RNAs and mRNAs, respectively.
T77 28677-28864 Epistemic_statement denotes Alternatively, a specific viral protein might be required to either bind the leader RNA to the RNA template or modify RNA polymerase to allow the transcription of body sequences of mRNAs.
T78 28865-28996 Epistemic_statement denotes Subunit regulation of a core polymerase has been proposed for the synthesis of alphavirus subgenomic mRNAs (Sawicki et al., 1978) .
T79 29261-29327 Epistemic_statement denotes However, these leader RNAs are not joined covalently to the mRNAs.
T80 29328-29582 Epistemic_statement denotes While the function of the bunyavirus leader RNAs has not been determined, the 47-nucleotide VSV leader is transported to the nucleus and serves as an inhibitor of host transcription (Grinell and Wagner, 1984; Kurilla et al.. 1982; McGowan et al., 1982) .
T81 29583-29652 Epistemic_statement denotes Similar functions have not been detected with coronavirus leader RNA.
T82 29750-29929 Epistemic_statement denotes Although influenza virus does not synthesize a free leader RNA, it requires a host-derived 5'-capped RNA as a primer for transcription (Bouloy et al., 1978; Plotch et al., 1981) .
T83 29930-30046 Epistemic_statement denotes The leader-primed mechanism for RNA transcription employed by coronaviruses might have even more general occurrence.
T84 30047-30259 Epistemic_statement denotes It has been shown that the mRNAs of the variable surface glycoprotein (VSG) in African trypanosomes contain an identical 37-nucleotide stretch at their 5'-ends (Boothroyd and Cross, 1982; Longacre et al., 1983) .
T85 30260-30427 Epistemic_statement denotes More recent studies have shown that a free leader RNA is synthesized which might serve as the primer for the VSG mRNA (Campbell et al., 1984; Milhausen et al., 1984) .
T86 30428-30571 Epistemic_statement denotes Thus, this leader-primed RNA transcription might represent a common alternative mechanism for generating spliced mRNAs in eukaryotic organisms.