Modulation of p53 activity by IκBα: Evidence suggesting a common phylogeny between NF-κB and p53 transcription factors

BACKGROUND: In this work we present evidence that the p53 tumor suppressor protein and NF-κB transcription factors could be related through common descent from a family of ancestral transcription factors regulating cellular proliferation and apoptosis. P53 is a homotetrameric transcription factor known to interact with the ankyrin protein 53BP2 (a fragment of the ASPP2 protein). NF-κB is also regulated by ankyrin proteins, the prototype of which is the IκB family. The DNA binding sequences of the two transcription factors are similar, sharing 8 out of 10 nucleotides. Interactions between the two proteins, both direct and indirect, have been noted previously and the two proteins play central roles in the control of proliferation and apoptosis. RESULTS: Using previously published structure data, we noted a significant degree of structural alignment between p53 and NF-κB p65. We also determined that IκBα and p53 bind in vitro through a specific interaction in part involving the DNA binding region of p53, or a region proximal to it, and the amino terminus of IκBα independently or cooperatively with the ankyrin 3 domain of IκBα In cotransfection experiments, κBα could significantly inhibit the transcriptional activity of p53. Inhibition of p53-mediated transcription was increased by deletion of the ankyrin 2, 4, or 5 domains of IκBα Co-precipitation experiments using the stably transfected ankyrin 5 deletion mutant of κBα and endogenous wild-type p53 further support the hypothesis that p53 and IκBα can physically interact in vivo. CONCLUSION: The aggregate results obtained using bacterially produced IκBα and p53 as well as reticulocyte lysate produced proteins suggest a correlation between in vitro co-precipitation in at least one of the systems and in vivo p53 inhibitory activity. These observations argue for a mechanism involving direct binding of IκBα to p53 in the inhibition of p53 transcriptional activity, analogous to the inhibition of NF-κB by κBα and p53 by 53BP2/ASPP2. These data furthermore suggest a role for ankyrin proteins in the regulation of p53 activity. Taken together, the NFκB and p53 proteins share similarities in structure, DNA binding sites and binding and regulation by ankyrin proteins in support of our hypothesis that the two proteins share common descent from an ancestral transcriptional factor

Cytoplasmic DROSHA and non-canonical mechanisms of MiR-155 biogenesis in FLT3-ITD acute myeloid leukemia

We report here on a novel pro-leukemogenic role of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) that interferes with microRNAs (miRNAs) biogenesis in acute myeloid leukemia (AML) blasts. We showed that FLT3-ITD interferes with the canonical biogenesis of intron-hosted miRNAs such as miR-126, by phosphorylating SPRED1 protein and inhibiting the "gatekeeper" Exportin 5 (XPO5)/RAN-GTP complex that regulates the nucleus-to-cytoplasm transport of pre-miRNAs for completion of maturation into mature miRNAs. Of note, despite the blockage of "canonical" miRNA biogenesis, miR-155 remains upregulated in FLT3-ITD+ AML blasts, suggesting activation of alternative mechanisms of miRNA biogenesis that circumvent the XPO5/RAN-GTP blockage. MiR-155, a BIC-155 long noncoding (lnc) RNA-hosted oncogenic miRNA, has previously been implicated in FLT3-ITD+ AML blast hyperproliferation. We showed that FLT3-ITD upregulates miR-155 by inhibiting DDX3X, a protein implicated in the splicing of lncRNAs, via p-AKT. Inhibition of DDX3X increases unspliced BIC-155 that is then shuttled by NXF1 from the nucleus to the cytoplasm, where it is processed into mature miR-155 by cytoplasmic DROSHA, thereby bypassing the XPO5/RAN-GTP blockage via "non-canonical" mechanisms of miRNA biogenesis

METTL3-Mediated m6A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL.SignificanceMETTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171

Supplementary Tables S1-5 from METTL3-Mediated m⁶A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL

Table S1 shows CLL sample information; Table S2 shows protein expression detected in CLL and normal B cells using TMT proteomics; Table S3 shows top 150 transcripts with highest m6A density in B cells; Table S4 shows sequences of shRNAs, sgRNAs, and primers used in our study; Table S5 shows cellular pathways enriched in our study.</p

Supplementary Figures S1-8 from METTL3-Mediated m⁶A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL

Fig S1. SF3B1 mutant CLL samples had pervasive changes in 3’ splice site. Fig S2. Omics analyses identify widespread post-transcriptional upregulation of splicing factors in CLL. Fig S3. Abundance of spliceosome complexes and RNA-binding proteins are associated with clinical outcomes in CLL. Fig S4. METTL3 is consistently upregulated along with differential m6A modification on transcripts of RNA splicing process in CLL. Fig S5. KO or pharmacological inhibition of METTL3 impacts cell growth. Fig S6. KO or pharmacological inhibition of METTL3 impacts apoptosis, cell cycle, and splicing factor abundance. Fig S7. Splicing factors are either direct or indirect targets of METTL3. Fig S8. Validation of association between m6A and splicing factor abundance using dCasRx-METTL3.</p