11 Matching Annotations
  1. Dec 2017
    1. Zika infection disrupts the binding of MSI1 to its endogenous targets

      Zika virus is dangerous for fetuses, but in adults the virus may eradicate cancerous brain tumors.

      Read more about the Zika virus uses for brain tumors here: https://medicine.wustl.edu/news/zika-virus-kills-brain-cancer-stem-cells/

    2. To this end, luciferase RNA flanked by the 5′ and 3′UTRs of PE243, was transfected with increasing amounts of MSI1 into human embryonic kidney (HEK) 293T cells, which do not normally express MSI1 (fig. S4).

      Luciferase RNA was transfected into human embryonic kidney cells with increasing amounts of MSI1 to test the translation regulation abilities of MSI1. The result is an increase in luciferase translation due to MSI1 regulation (meaning MSI1 may stabilize the genome of RNA).

    3. Because there was no discernible difference between ZIKV binding and entry into control and KO cells (Fig. 2, G and H), we asked if MSI1 could regulate translation through ZIKV UTRs

      The viral binding affinity was tested using the viral binding assay along with the a pseudotyped particle infectivity assay to test the ability of the virus to infect a cell. The results for the assays did not show a significant change in binding affinity.

    4. Consistently, levels of the viral dsRNA and flavivirus E protein, as well as the infectious titer, were reduced in the KOs (Fig. 2E and fig. S3). Because MSI2 levels were similar between control and KO cells (Fig. 2C), MSI1 and MSI2 are unlikely to have complete functional redundancy in ZIKV replication. Replication of the MR766 strain was also impaired in the KO cells (Fig. 2F)

      Numbers of viral dsRNA, flavivirus E protein, and infectious particles were determined using confocal microscopy. Copies of viral RNA were measured after introducing the MR766 strain. The authors use this method to understand the replication processes that can increase or, in this article, decrease due to the inactivatoin of MSI1. The results observed showed a decrease in the amounts of viral particles and MR766 replication.

    5. We then generated MSI1 knockouts (KOs) in U-251 cells by clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9–mediated targeting of exons 8 or 6 of MSI1 (KO1 and KO2, respectively; Fig. 2C and fig. S2). Control cells were obtained through clonal expansion of cells transfected with Cas9 alone. By measuring viral RNA at different times after PE243 infection, a marked reduction of viral load was seen in KO1 and KO2 cells at 24 and 48 hours (Fig. 2D).

      MSI1 knockout cells (KO) were created using CRISPR/Cas9. KO1 and KO2 were then probed with N and C termini MSI1 and MSI2 antibodies. The results showed a decrease in viral load after 24 and 48 hours.

    6. In all three cell types, MSI1 depletion led to a marked reduction in viral RNA levels (Fig. 2, A and B)

      Cells were observed after being infected with PE243, a control siRNA (small interfering RNA used to silence protein coding genes), and MSI1 siRNA. Western blots were run on the proteins from the cells. The results of the observations showed a decrease in viral RNA levels.

    7. To investigate whether MSI1 also binds ZIKV 3′UTR in vivo, ultraviolet (UV) cross-linking immunoprecipitation (CLIP) of RNA was performed from lysates of PE243-infected U-251 glioblastoma cells, revealing a robust direct interaction between MSI1 and PE243 ZIKV RNA (Fig. 1E)

      UV radiation was used to run a CLIP (cross-linking immunoprecipitation used to bind proteins together using UV radiation) analysis on infected cells. A western blot was run on the results, which allowed scientists to find the strong bond between MSI1 and PE243 ZIKV RNA.

    8. By mapping these sites onto a predicted secondary structure of ZIKV 3′UTR, we found all three sites to be present on stem-loop structures, which are considered optimal for MSI binding

      Uren et al., (2015) investigated the mechanism of action of the RNA-binding protein MSI1 to help identify target genes. Using individual-nucleotide resolution cross-linking and immunoprecipitation, researchers found that MSI1 prefers UAG sequences with a specific structure, especially in the 3’ untranslated region. MSI1 target RNAs encode proteins involved in cell proliferation and adhesion, known to underlie the spread of glioblastomas.

    9. analysis of the genomic RNA of the Brazilian ZIKV strain, PE243

      Donald and colleagues (2016) researched the genomic sequences of the zika virus to understand the host and viral interactions along with the process of replication. One response, interferon, to the zika virus is suppressed to allow the virus to replicate. siRNA causes the suppression of the interferon response.

    10. Consistently, in ZIKV-infected cells, MSI1 colocalized with double-stranded RNA (dsRNA), a viral replication intermediate, as visualized by confocal and stimulated emission depletion (STED) super-resolution microscopy (Fig. 1, F and G)

      An immunofluorescence assay run of PE243 infected cells allowed scientists to detect MSI1, dsRNA and DNA with the use of confocal microscopy and STED (uses fluorescence to overcome any diffraction that may be seen in the confocal microscopy) super resolution microscopy. The results were finding MSI1 and dsRNA near each other in the cell.

    11. Two sites were conserved between PE243 and the Ugandan MR766 strains (sites 1 and 2), whereas the third (site 3) was found only in the Asian-lineage strains including PE243 (Fig. 1A and fig. S1, A and B)

      The 3' UTR (untranslated region) of PE243 (the Brazilian ZIKV strain) was used in an RNA pull-down assay. The biotinylated (covalently bound to biotin) PE2443 RNA was incubated with cell extracts. Finally, a Western blot was run to conclude the two conserved with the Ugandan MR677 strain and one with the Asian strain.