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. 2022 Aug 1:13:939863.
doi: 10.3389/fimmu.2022.939863. eCollection 2022.

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing

Mariam Shallak  1 , Tiziana Alberio  2 , Mauro Fasano  2 , Maria Monti  3   4 , Ilaria Iacobucci  3   4 , Julien Ladet  5 , Franck Mortreux  5 , Roberto S Accolla  1 , Greta Forlani  1
Affiliations

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing

Mariam Shallak et al. Front Immunol. .

Abstract

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.

Keywords: ATL; HBZ; HTLV-1; alternative splicing; interactome; protein network.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Identification of potential HBZ interactors. Schematic workflow of the proteomic analysis performed in this study (graphics using BioRender.com).
Figure 2
Figure 2
HBZ protein interaction network. (A) Graphic representation of endogenous HBZ interacting nuclear factors in the ATL-2 leukemic cells. (B) Reciprocal protein-protein interaction network of the 249 HBZ interactors. (C) Subnetworks from HBZ interactors network clustered by the GLay algorithm: RNA splicing sub-cluster (dark blue), JAK/STAT signaling pathway sub-cluster (dark grey), non-mediated mRNA decay (NMD) sub-cluster (violet).
Figure 3
Figure 3
Comparative transcriptome analysis of HTLV-1-derived ATL-2 vs non HTLV-1-derived MOLT-4 leukemic cells, and of Jurkat-HBZ vs Jurkat cells. (A) Overview of the strategy used to identify the changes in both the cellular transcriptome and alternative splicing landscape associated with HTLV-1-derived ATL-2 cells or with Jurkat-HBZ transfected cells. (B) Volcano plot of significantly up-regulated genes (red dots) and down-regulated genes (blue dots) with |log2fold-change|>1.0 (p < 0.05) in ATL-2 vs MOLT-4 cells (left panel) or Jurkat-HBZ expressing cells vs Jurkat cells (right panel). Dark dots represent the dysregulated genes with 0.4<|log2fold-change|<1.0 (p < 0.05). (C) Venn diagram showing differentially expressed genes (DEG) that are shared between ATL-2 and Jurkat-HBZ expressing cells.
Figure 4
Figure 4
Alternative splicing modifications upon HTLV-1-derived leukemic transformation or HBZ expression. (A) Schematic representation of five main alternative splicing events (lines in red), listed in the right. (B) Alternative Splicing Events (ASEs) detected in ATL-2 (left) and Jurkat-HBZ (right) cells. (C) Exclusion (deltaPSI<0) and Inclusion (deltaPSI>0) number of ASEs witnessed in ATL-2 (left) and Jurkat-HBZ (right) cells are indicated within the columns of the bar graph.
Figure 5
Figure 5
Comparative analysis between ATL-2 and Jurkat-HBZ cells. (A) Venn diagrams showing differentially expressed and alternatively spliced genes that are shared between ATL- 2 (left) and Jurkat-HBZ (right) cells. (B) Donut pie representing the number of genes undergoing ASEs shared between ATL-2 and Jurkat-HBZ cells. (C) List of shared cancer related genes alternatively spliced in ATL-2 and Jurkat-HBZ cells. (D) RT-PCR validation of microarray-predicted exon events in ATL-2 as compared to MOLT-4 and healthy PBMCs, and in Jurkat-HBZ cells compared to Jurkat cells. RNA seq derived delta PSI values are indicated below each PCR reaction. Gene designation is on the left and amplification boundaries are on the right with numbers representing the specific exons. The arrows represent the position of the designed primers.
Figure 6
Figure 6
Shared target exons between HBZ and HBZ-interacting splicing factors. (A) 32 different splicing factors interacting with HBZ are shown with inside the numbers of target exons they share with HBZ target exons. (B) Venn diagram showing the number of inter-common target exons between HBZ and each of the major splicing factors: DDX5/17, SRSF1, U2AF1, U2AF2, and HNRNPC.
Figure 7
Figure 7
In vivo interaction and colocalization of endogenous HBZ with DDX5 and DDX17 in ATL-2 cells. (A) HBZ interaction with DDX5 and DDX17 in ATL-2 cells was assessed by co-immunoprecipitation assay. Nuclear and cytoplasmic protein extracts of Jurkat and ATL-2 cells were immunoprecipitated with the anti-HBZ 4D4-F3 mAb (IP HBZ) and the eluted material analyzed by western blotting for the presence of co-immunoprecipitated DDX5 and DDX17 molecules using specific monoclonal antibodies. Input levels of respective DDX5 and DDX17 are represented in the lower panels. (B) The purity of nuclear and cytoplasmic fraction extracted fractions was verified by western blot (input) using ten percent of the nuclear and cytoplasmic protein extracts and antibodies specific for the nuclear protein Nup98 or β-tubulin for cytoplasmic protein. N, nuclear; C, cytoplasmic. ATL-2 cells were reacted in a pairwise combination with the 4D4-F3 anti-HBZ mAb and either polyclonal rabbit anti-DDX5 (C) or anti-DDX17 (D) antibodies. Anti-HBZ mAb was revealed by Alexa fluor 546-labeled goat anti-mouse IgG (red), whereas the other rabbit antibodies were revealed by Alexa fluor 488-labeled goat anti-rabbit antibodies (green). The colocalization is represented in the overlay panels (yellow). The region of interest (ROI) drawn along mid-nucleus level of the merge image is represented by red (HBZ) and green (RNA helicases) peaks in the histogram. At least 200 cells were analyzed. All scale bars are 5 µm.
Figure 8
Figure 8
In vivo interaction and colocalization of exogenous HBZ with DDX5 and DDX17 in Jurkat-HBZ cells. (A) HBZ interaction with DDX5 and DDX17 in Jurkat-HBZ cells was assessed by co-immunoprecipitation assay. Nuclear and cytoplasmic protein extracts of Jurkat and Jurkat-HBZ cells were immunoprecipitated with anti-HBZ 4D4-F3 mAb (IP HBZ) and the eluted material was analyzed by western blotting for the presence of co-immunoprecipitated DDX5 and DDX17 molecules using specific monoclonal antibodies. Input levels of respective HBZ, DDX5 and DDX17 are represented in the lower panels. (B) The purity of nuclear and cytoplasmic extracted fractions was verified by western blot (input) using ten percent of the nuclear and cytoplasmic protein extracts and antibodies specific for the nuclear protein Nup98 or β-tubulin for cytoplasmic protein. N, nuclear; C, cytoplasmic. Jurkat-HBZ expressing cells were reacted in a pairwise combination with the 4D4-F3 anti HBZ mAb antibody, and either polyclonal rabbit anti-DDX5 (C) or anti-DDX17 (D) antibodies. Anti-HBZ mAb was revealed by Alexa fluor 546-labeled goat anti-mouse IgG (red), whereas the other rabbit antibodies were revealed by Alexa fluor 488-labeled goat anti-rabbit antibodies (green). The colocalization is represented in the overlay panels (yellow). The region of interest (ROI) drawn along mid-nucleus level of the merge image is represented by red (HBZ) and green (RNA helicases) peaks in the histogram. At least 300 cells were analyzed. All scale bars are 5 µm.
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