Long-term reduction of T-cell intracellular antigens leads to increased beta-actin expression.
BACKGROUND
The permanent down-regulated expression of T-cell intracellular antigen (TIA) proteins in HeLa cells improves cytoskeleton-mediated functions such as cell proliferation and tumor growth.


METHODS
Making use of human and mouse cells with knocked down/out expression of T-cell intracellular antigen 1 (TIA1) and/or TIA1 related/like (TIAR/TIAL1) proteins and classical RNA (e.g. reverse transcription-quantitative polymerase chain reaction, polysomal profiling analysis using sucrose gradients, immunoblotting, immunoprecipitation, electrophoretic mobility shift assays, ultraviolet light crosslinking and poly (A+) test analysis) and cellular (e.g. immunofluorescence microscopy and quimeric mRNA transfections) biology methods, we have analyzed the regulatory role of TIA proteins in the post-transcriptional modulation of beta-actin (ACTB) mRNA.


RESULTS
Our observations show that the acquisition of above cellular capacities is concomitant with increased expression levels of the actin beta subunit (ACTB) protein. Regulating TIA abundance does not modify ACTB mRNA levels, however, an increase of ACTB mRNA translation is observed. This regulatory capacity of TIA proteins is linked to the ACTB mRNA 3'-untranslated region (3'-UTR), where these proteins could function as RNA binding proteins. The expression of GFP from a chimeric reporter containing human ΑCΤΒ 3'-UTR recapitulates the translational control found by the endogenous ACTB mRNA in the absence of TIA proteins. Additionally, murine embryonic fibroblasts (MEF) knocked out for TIA1 rise mouse ACTB protein expression compared to the controls. Once again steady-state levels of mouse ACTB mRNA remained unchanged.


CONCLUSIONS
Collectively, these results suggest that TIA proteins can function as long-term regulators of the ACTB mRNA metabolism in mouse and human cells.
Isabel
I
Carrascoso
Carmen
C
Sánchez-Jiménez
Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid (CSIC/UAM), C/Nicolás Cabrera 1, Cantoblanco, DP 28049 Madrid, Spain. jmizquierdo@cbm.uam.es.
José M
JM
Izquierdo
D020413:3' Untranslated Regions; D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D039102:Poly(A)-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D000074019:T-Cell Intracellular Antigen-1
D020413:3' Untranslated Regions; D000199:Actins; D039102:Poly(A)-Binding Proteins; D016601:RNA-Binding Proteins; D000074019:T-Cell Intracellular Antigen-1; C071558:TIA1 protein, human; C499367:Tia1 protein, mouse; C489665:Tial1 protein, mouse; D049452:Green Fluorescent Proteins; C076751:TIAL1 protein, human