IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.
TheIBTKgene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and afterIBTKknock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed inIBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed inIBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated byIBTKsilencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition,IBTKRNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate thatIBTKdifferently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. fiume@unicz.it.
Giuseppe
G
Fiume
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. annarita.scialdone@med.lu.se.
Annarita
A
Scialdone
Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Schola Medica Salernitana', University of Salerno, via S. Allende 1, 84081 Baronissi, Italy. frizzo@unisa.it.
Francesca
F
Rizzo
Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Schola Medica Salernitana', University of Salerno, via S. Allende 1, 84081 Baronissi, Italy. mdefilippo@unisa.it.
Maria Rosaria
MR
De Filippo
Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Schola Medica Salernitana', University of Salerno, via S. Allende 1, 84081 Baronissi, Italy. laudanna@unicz.it.
Carmelo
C
Laudanna
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. albano@unicz.it.
Francesco
F
Albano
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. golino@unicz.it.
Gaetanina
G
Golino
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. eleonoravecchio@unicz.it.
Eleonora
E
Vecchio
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. pontoriero@unicz.it.
Marilena
M
Pontoriero
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. mimmi@unicz.it.
Selena
S
Mimmi
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. ceglia@unicz.it.
Simona
S
Ceglia
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. pisano@unicz.it.
Antonio
A
Pisano
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. iaccino@unicz.it.
Enrico
E
Iaccino
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. cpalmieri@unicz.it.
Camillo
C
Palmieri
Department of Health Sciences, University "Magna Graecia", 88100 Catanzaro, Italy. paduano@unicz.it.
Sergio
S
Paduano
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. viglietto@unicz.it.
Giuseppe
G
Viglietto
Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Schola Medica Salernitana', University of Salerno, via S. Allende 1, 84081 Baronissi, Italy. aweisz@unisa.it.
Alessandro
A
Weisz
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. scala@unicz.it.
Giuseppe
G
Scala
Department of Experimental and Clinical Medicine, University 'Magna Graecia' of Catanzaro, Viale Europa (Località Germaneto), 88100 Catanzaro, Italy. quinto@unicz.it.
Ileana
I
Quinto
D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002465:Cell Movement; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020014:K562 Cells; D009707:Nucleosomes / Q000378:metabolism / Q000648:ultrastructure; D009928:Organ Specificity; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D014176:Protein Biosynthesis; D000072417:Protein Domains; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059467:Transcriptome; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; C437966:IBtk protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009707:Nucleosomes; C114487:PDCD4 protein, human; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex
Cul3-dependent E3 ligase; IBTK; Next Generation Sequencing; transcription