Gottweib (German Edition)

We present multiple lines of evidence that these miRNA: We also tested the functionality of miRNA: With the exception of a viral miRNA with poor targeting proficiency Garcia et al. Computational analyses of our chimera-identified miRNA: Our data further suggest that mismatches in the seed occur predominantly at positions 2 and 7. These data allow insights into principles by which miRNA recognize target sites. Briefly, worms incorporated photoreactive 4-thiouridine nucleosides 4sU into their RNA, which crosslinks to bound proteins during UV irradiation.

After homogenization, the lysate was treated with RNase T1. After homogenization of worms, the lysate is treated with RNase T1. Some miRNAs are shortened, others remain complete. Crosslinked RNA is recovered and deep sequenced. B Example of a miRNA interaction recovered from chimeric reads. Predicted reconstruction of the miRNA: T to C conversion.

A comparable fraction of chimeras with truncated miRNAs was also found in a control sample, to which no ligase was added to generate chimeras. D miRNA and target ends involved in the ligations of the control sample are highly enriched in an upstream G, suggesting that RNase T1 generated the ends used for this type of ligation.

See also Figure S1. Control samples were generated without the addition of a ligase. This frequency was very high Our bioinformatics analyses Methods revealed the presence of thousands of miRNA-chimeric reads in the ligation samples Figure 1B. Consequently, we mapped chimera target sequences directly to AGO sites.

This increased the sensitivity of target recovery due to the smaller search space Methods. In total, we identified 3, miRNA: While ligation samples had miRNA: RNase T1 cuts with high preference after guanines, strongly suggesting that RNase T1 produced the ends used as substrates for this ligation reaction. Bacteria are the food source for C. We screened for perfect non G: U complementarity to miRNA nts seed , complementarity to miRNA nts containing one mismatched or bulged nucleotide and complementarity to miRNA nts containing two mismatches.

Shuffled sequences dinucleotides in target sequences are permuted served as control. B Hybridization profile summarized over all interactions. Duplex structures of miRNA: Shuffled sequences dinucleotides in target sequences are permuted and shuffled interactions targets are swapped between miRNAs served as control. Shuffling target sites between miRNA families served as control. D Local frequency of crosslink-induced T to C conversions in target RNAs from interactions with a perfect seed match normalized to local thymidine frequency.

Nucleotides hybridized to the seed of the miRNA are strongly indisposed to crosslink with the protein. See also Figure S2. The median free energy was lower by 3. Base pairing was as expected for miRNA Wee et al. As for interactions with perfect seed matches, analysis of miRNA: However, their binding free energy was less decreased Figure S2E and hybridization profiles did not indicate enriched base pairing within the seed region Figure S2F. Since miRNA family members have the same seed, they are expected to share some of their targets and indeed, target sites were much more often ligated to members of the same miRNA family than expected by chance.

This is because noncrosslinked miRNAs tend to be lost under the denaturing conditions of protein purification, while ligated, noncrosslinked miRNAs can pass purification due to their covalent connection to AGO. If in our C. See also Table S3. Mismatches were broadly distributed over all types of nucleotides, including G: C Individual target sites are more often ligated to members of the same miRNA family than expected by chance. Positions hybridized to the seed of the miRNA are strongly indisposed to crosslink. E Mismatches in seed sites occur predominantly at position 2 or 7 of the miRNA.

Shown is the positional mismatch frequency for interactions with a match containing 1 mismatch, averaged over different miRNA families. See also Figure S3 and Table S1. The large numbers of miRNA: Position 2 and 7 of the miRNA showed a significantly increased frequency of mismatches than the positions in between. This pattern was conserved across human, mouse, and C. Interesting examples include miRa and miRb, which differ only by one nucleotide at position Our results show partial agreement with the findings of Helwak et al.

Our analysis of human miRNA chimeras in data by Kishore et al. We analyzed the overlap between miRNA: We recorded how often the miRNA that we had found ligated to a site corresponded to the top-ranking miRNA predicted for this site. These results highlight the value of unambiguous identification of imperfect seed binding by biochemical methods.

First evidence that chimera-identified miRNA: For instance, in C. We next asked whether chimera-identified miRNA: We searched these data for chimeras and discovered 46 miR interactions in the WT sample and, as expected, none in the miR KO sample. C and changes in protein abundance after overexpression of miR in a human cell line Selbach et al.

E and F Conservation across 31 vertebrate species of perfect seed 2—7 matches E and seed matches with 1 nt mismatch 1 mm F from human miRNA: Conservation of other seed matches for the same miRNA served as a control. A perfect seed match in human was counted as conserved if present at the same position in the alignment. A seed match with 1mm was deemed conserved if the identical 1mm seed match or the perfect seed match was present at the same position in the alignment.

See also Figure S4 and Table S2. To study functionality of our miRNA: A similar experiment was performed in human embryonic stem cells for the miR family and miR Lipchina et al. The majority of these interactions out of for data by Hafner et al. To assay the impact of miRNA: Indeed, protein synthesis for 91 miR targets identified by chimera analysis was highly significantly downregulated Figure 4D.

Finally, we analyzed evolutionary conservation of miRNA: It should be noted that the matches containing one mismatched nucleotide have a lower degree of conservation than perfect matches, which also holds true for C. Taken together, analysis of miRNA perturbation experiments and of target site conservation provides evidence that our miRNA: However, we decided to experimentally test the functionality of identified miRNA: For most viral miRNAs, target identification is severely complicated by the fact that these viruses encode mostly evolutionary novel and unique miRNAs and infect only humans.

Therefore viral miRNAs are not expected to preferentially bind conserved sites. The use of computational target prediction is difficult in this case. Moreover, the seeds of viral and human miRNAs can be similar, which causes difficulties in unambiguously assigning the targeting miRNA to a site. Previous studies have suggested that these viral miRNAs can regulate the same binding sites as the corresponding host miRNA and therefore function to mimic the host miRNA Gottwein et al.

The most prominent example is KSHV miR-K11, which shares sequence identity of nts with cellular miR and is known to mimic the oncogenic properties of miR in B cells Boss et al. To investigate whether chimera-discovered viral miRNA: All sites that mediated reporter repression by miR-K11 were also responsive to miR Figure S5B , suggesting that nts of miR-K11 are important for the observed regulation.

The importance of the miR-K11 seed, also for the noncanonical interactions, is further highlighted by the abrogation of regulation when the seed region was disrupted by a two nucleotide substitution Figure S5C. Thus, analysis of chimeric reads enabled us to identify functional canonical and noncanonical interactions. A The majority of tested, chimera -identified KSHV miR-K11 interactions resulted in specific reporter repression, including sites with weak seed matches.

Noncanonical interactions are marked with a diamond. B Predicted base pairing for noncanonical miR-K11 sites that were responsive in the reporter assay. Duplex structures were predicted by RNAhybrid, G: U allowed, controls permutation of dinucleotides in target sequences were subtracted. E Predicted base pairing of tested interactions at nonconserved sites; lack of conservation in seed matches shown for mouse, dog, chicken continued in Figure S5F.

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See also Figure S5. The situation becomes even trickier in the context of a viral infection, when either cellular or viral miRNAs may mediate targeting. The analysis of chimeric reads can therefore eliminate ambiguities in the assignment of the targeting miRNA s. We therefore examined chimera-identified miRK3: We selected 5 noncanonical miR-K3 interactions for further examination of their regulatory potential in reporter assays, but did not detect reporter repression mediated by any of these sites Figure S5E.

Thus, even chimera-identified binding sites for the special case of miR-K3 appear to have poor regulatory capacity, at least in the experimental system employed here. An important difficulty when investigating viral miRNAs with evolutionary novel seed sequences, i. Six interactions, including 5 for which the seed match was not conserved Figure 5E and Figure S5F , were tested for reporter repression. Leaving the exceptional case of miR-K3 aside, our results strongly support the notion that the analysis of miRNA: Our initial key aim was to ligate miRNAs to their target sites in order to identify these interactions from sequencing data.

We show for the model system C. Our data and analyses demonstrate that ligation reactions seem to occur specifically between miRNAs and bound targets.

βパナソニック 照明器具【NNY22210C】ＬＥＤガーデンライト昼白色タイプＨ８００ オンライン {L}：クローバー資材館 ☆☆NNY22210C

The reasons for this are a almost all target sites within chimeras have the crosslink specific T-C conversion b although bacterial RNAs from E. However, to our surprise we discovered that chimeras were also present in experiments in which we did not perform the additional ligation step via the T4 RNA ligase. The exact set of factors needed, however, and the mechanism of their action are not understood. A function of ubiquitin ligases in retrovirus budding is supported by the observations that a small percentage of Gag proteins within purified HIV and murine leukemia virus MLV is monoubiquitinated 19 , 20 and that large amounts of free ubiquitin are present inside retroviruses 19 , In addition, the intracellular depletion of free ubiquitin by treatment with proteasome inhibitors has been shown to block release of HIV-1, HIV-2, and Rous sarcoma virus RSV at a very late stage 22 , 29 , In the case of RSV, this block could be partially overcome by fusing ubiquitin to the C terminus of Gag It is presently not known if the machinery needed to separate viral and cellular membranes is identical or at least overlapping for viruses containing different L domains.

Different L-domain motifs have been shown to be at least partially exchangeable between retroviruses 17 , 21 , Viruses that contain a mutated PSAP motif were defective at a late stage of release, since late budding structures and chained procapsids accumulated at the cell surface. Hunter [ 28 ]. All mutations were introduced by standard PCR mutagenesis, and amplified fragments were sequenced.

The region between the pcDNA3. Detailed cloning procedures are available on request. All other transfections were carried out by the calcium phosphate precipitation method. The pulse period was ended by adjusting the medium to 1 mM unlabeled methionine Sigma , followed by removal of the labeling medium.

Aliquots were chased in DMEM containing 0. Thirty hours posttransfection, cells were rinsed with PBS and incubated with [ 35 S]methionine 0. Twelve hours after transfection, cells were rinsed with PBS, and the medium was replaced. Virus-containing medium was harvested 36 h after transfection and filtered through a nm-pore-size filter. The visible virus band was isolated and diluted with PBS. Purity of the preparation was monitored by silver staining and evaluated based on the loss of albumin.

For external digestion of viral particles with trypsin, sucrose pellets were resuspended in 0. When virus was lysed with 0. Western blotting was performed by using standard procedures. The following primary antibodies were used: At 48 h after transfection, cells were gently rinsed with PBS and fixed with ice cold 2. Micrographs were taken with a Zeiss EM electron microscope at 80 kV. The magnification indicator was routinely controlled by the use of a grating replica. The same pattern of Gag and CA proteins was detected in both cases Fig.

The Mason-Pfizer Monkey Virus PPPY and PSAP Motifs Both Contribute to Virus Release

In agreement with previously published results, intracellular processing of Gag was observed for both constructs 28 , The extent of intracellular processing did not correlate with the relative level of Gag expression. In addition, we constructed plasmids containing both mutations Fig. The amino acid substitutions were chosen based on published data 12 , 17 , 34 and should result in complete inactivation of the respective motif.

To determine the efficiency of wt and mutant virus release, HeLaP4 cells were transfected with the indicated plasmids and, 36 h after transfection, metabolically labeled with [ 35 S]methionine for 12 h. After the labeling period, cells were lysed. Virus was collected by centrifugation through sucrose cushions. The result of a representative experiment is shown in Fig.

Peter Bauer-Gottwein - Journals - DTU Orbit

Transfection of wt pMPMV yielded the typical pattern of cell-associated Gag and CA proteins and caused the release of virus into the growth medium Fig. It should be noted that all virus samples contained only completely processed CA and no precursor or intermediate processing products, independent of the processing defect observed in transfected cells data not shown. Mutation of the PPPY motif resulted in a pronounced impairment of intracellular Gag processing and a defect in particle release Fig. Analysis of the virus fraction Fig.

This result is in agreement with the report by Yasuda et al. Analysis of PSAP mutant-transfected cells showed a slight impairment of processing and a significant reduction in virus release Fig. Cells were transfected with the indicated pMPMV constructs and metabolically labeled with [ 35 S]methionine for 12 h. Note that no precursor or intermediate processing product was observed in the virus samples.

CA-containing bands were quantified. The release efficiency of wt and mutant virus as well as that of mixed virus populations was calculated by dividing the signal from virus-associated CA protein by the sum of the signals from cell- and virus-associated CA-containing proteins. The obtained numbers were normalized for wt release efficiency.

Cells were cotransfected with the indicated pSHRM15 plasmids, and cell- and virus-associated viral proteins upper and lower panel, respectively were analyzed by Western blotting with an anti-CA antiserum. Again, no precursor or intermediate processing product was observed in the virus samples. It has been suggested that the magnitude of the defect in virus release caused by L-domain mutations may correlate with the level of Gag expression The described experiments were performed with a CMV-driven high-level expression plasmid transfected by lipofection. To exclude the possibility that the intermediate phenotype of the PSAP mutant was a result of high levels of expression, we repeated the experiment by using transfection of pSHRM15 into HeLaP4 cells by calcium phosphate precipitation Fig.

Accordingly, the intermediate phenotype of the PSAP mutant is not due to overexpression. To quantify the relative efficiencies of wt and mutant virus release from pMPMV-transfected cells, the virus-associated CA signal was related to the sum of the signals of the intracellular and virus-associated CA-containing proteins. Only the three major CA-containing bands namely, Gag, the protein migrating slightly above Gag, and processed CA detected in cell extracts were quantified. The obtained numbers were normalized for the methionine content of the respective proteins.

The protein migrating above Gag was assumed to carry the same number of methionines as Gag.

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It has been reported that RSV L-domain mutant Gag protein can be rescued into virus-like particles by coexpression of Gag protein carrying an intact L domain These results suggest that only a minor fraction of Gag molecules of a budding virion needs to carry an L domain. To test the release efficiency of mixed populations of MPMV Gag proteins, we cotransfected equal amounts of mutant and wt plasmids and analyzed and quantified CA-reactive proteins in cell and virus extracts 24 h post transfection Fig. A similar analysis for the PSAP motif mutant was not conclusive, because differences in virus release were too small Fig.

The experiments shown in the previous section suggested that both motifs contribute to MPMV release. To test whether the PPPY and PSAP motifs can complement each other in trans or need to be present on the same polyprotein, we performed cotransfection experiments. Thus, both motifs can complement each other in trans under low- and high-expression conditions. As noted above Fig. When wt and mutant plasmids were cotransfected at a ratio of 1: Cotransfection of wt and PPPY mutant plasmids also did not restore intracellular Gag processing, since only Gag and the protein slightly above Gag were detected in cell lysates Fig.

The dominant-negative effect of the PPPY mutant Gag protein was also evident when high levels of expression were used Fig. In addition, we confirmed our finding by quantitative Western blotting data not shown. Taken together, these results suggest that a molar excess of PPPY mutant Gag polyproteins over wt Gag has a dominant-negative influence on virus production. In both cases, no dominant-negative effect on virus release was observed data not shown. Steady-state levels of cell- and virus-associated CA protein upper and lower panel, respectively were analyzed by Western blotting with anti-CA antiserum.

As shown above, intracellular processing of the PPPY and double-mutant Gag polyproteins was significantly impaired Fig. To analyze the processing kinetics of wt and mutant Gag proteins and to determine whether the protein migrating slightly above Gag represents a posttranslational modification of Gag, we performed pulse-chase experiments. Twenty-four hours after transfection, cells were pulse-labeled with [ 35 S]methionine for 30 min and chased for the times indicated.

Cell and virus extracts were immunoprecipitated with anti-CA antibodies. Directly after the pulse, the Gag polyprotein was the predominant band detected lane 1 , and processing to CA became clearly detectable after a 1-h chase lane 3. Most of the intracellular Gag was processed to CA after 8 h. Release of virus particles containing processed CA was detected after 1 h and increased over the entire chase period.

Kinetic analysis of wt and mutant Gag processing. Cells were chased in DMEM for the indicated time. HeLaP4 cells were transfected with the indicated pMPMV constructs, labeled as described above, and chased for 0, 2, or 8 h. At the 2-h time point, a Gag-related band arrow that migrates slower than Gag was detected in cells expressing PPPY mutant and double mutant Gag. A comparison of wt and mutant Gag processing kinetics is shown in Fig. Similar amounts of Gag polyproteins were observed in all cases after the pulse period, and the protein migrating above Gag was not detected in these samples Fig.

Furthermore, the protein migrating slightly slower than Gag marked by an arrow in lanes 6, 8, 10, and 12 became detectable in cells transfected with the PPPY or double mutant after a chase period of 2 h and remained present after 8 h. Therefore, this product corresponds to a posttranslationally modified version of Gag. We hypothesize that it represents Gag lacking the proline-rich C-terminal p4 domain Fig.

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CA-containing cleavage products migrating slower than their precursors have been described for other retroviruses Alternatively, the product migrating just above Gag could result from a posttranslational modification of Gag other than processing. Analysis of particle formation by EM is a more direct way to determine virus release at the morphological level. In all cases, the typical sites of intracellular assembly of immature procapsids were observed shown representatively for cells transfected with wt pMPMV in Fig.

Furthermore, thin sections of cells transfected with the wt plasmid contained many procapsids budding from the plasma membrane as well as numerous mature and immature virions Fig. Instead, many immature procapsids accumulated underneath the plasma membrane and appeared to be arrested at an early stage of bud formation Fig.

Typical late budding structures connected to the plasma membrane by a thin membrane stalk as well as chained budding structures containing more than one procapsid were also present, indicating that the early budding arrest was overcome by some procapsids Fig. Interestingly, in several cells, procapsids were found to decorate intracellular vesicles Fig.



Again, the majority of these procapsids failed to initiate bud formation, and no bulging of the membrane was observed. Bars, nm a and nm b. In this case, we observed predominantly late immature budding structures, many of which were tethered to each other Fig.

Some of these budding chains containing multiple procapsids were apparently released from the cell Fig. Less extracellular particles were observed than in the case of wt pMPMV, but some mature virions containing a condensed core were detected e. On the other hand, the relative abundance of budding structures and virus chains was increased in the case of the PSAP variant. Nearly one-half of the chains containing multiple procapsids were apparently released from the cell, i.

The relative abundance of immature procapsids was also significantly higher for the PSAP variant, indicating that maturation is impaired in this case. Taken together, these results showed that the PSAP mutant is indeed defective at the stage of virus release.

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Interestingly, the budding arrest in the case of the PSAP variant appeared to be at a later stage than in the PPPY variant, with more advanced budding structures extending further away from the plane of the plasma membrane. As in the biochemical experiments described above, the double mutant was indistinguishable from the PPPY mutant at the morphological level as well. Bars, nm a and nm b to d. Accordingly, the cellular binding partners should be recruited to the budding site and may be incorporated into virions.

The overexpression of a segment of TSG by transfection led to incorporation of this protein into HIV-1 particles 2. Virus samples were further purified by velocity gradient centrifugation as described previously 4.