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Physiopathology
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User name	Arndt Benecke
Email	arndt@ihes.fr
Phone / Fax number	+33160926665 / +33160926609
Location	Institut des Hautes Etudes Scientifiques (Integrative Biology) - 35, route de Chartres - 91440 Bures sur Yvette, France

Scientific description

The TAF6Î´ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6Î´ is an inducible a pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6Î´ has been shown to be a pivotal event in triggering death via the TAF6Î´ pathway, yet the nothing is currently known about the mechanisms that promote TAF6Î´ splicing.
Furthermore the transcriptome impact of the gain of function of TAF6Î´ function versus the loss of function of the major TAF6Î´ splice form remain undefined. Here we employ
comparative microarray analysis to show that TAF6Î´ drives a transcriptome profile distinct from that resulting from depletion of TAF6Î´. To define the cis-acting RNA elements responsible for TAF6Î´ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6Î´ and also reveal a role for RNA secondary structure in the selection of TAF6Î´.

Technical description

Hela ws cell line was maintained in culture in Dulbecco's modified Eagle's medium
supplemented with 2.5% fetal calf serum and 2.5% calf serum.
siRNA 5'-rGrGrArGrUrGrUrCrCrArGrArArGrUrArCrArUrCrGrUrGGT-3' (T6-1) and 5'-
rCrGrCrUrArArGrCrGrGrArArGrGrArArGrUrUrGrUrArGAT-3' (T6-2) were employed to
deplete all known splice variants of TAF6. SiRNA were transfected at a final concentration of 10nM with lipofectamine 2000 (Invitrogen) as a delivery agent (1.6 Î¼l/ml) according to the manufacturerâ€™s recommendations. 250 to 300ng of wild-type or mutant TAF6 minigene were transfected with 1Î¼l DMRIE-C (Invitrogen) per well in 24 well plate according to the manufacturerâ€™s recommendations. Cells were transfected by siRNA T6-1 two times. The second transfection was performed 24h after the first one and culture was maintained for a total of 48 or 72h before harvesting cells for RNA extraction (RNeasy Qiagen) for microarray analysis or 64h for protein analysis. All transfections were performed with OptiMEM medium (Invitrogen). Each transfection experiment was repeated three times.
RNA isolation and quality control were performed as previously described [2,22]. Applied
Biosystems HGS V2 arrays [23] were hybridized, washed, and exposed according to the
protocols by the technology provider. Raw data were quality controlled [24], and normalized using NeONORM (k-parameter=0.2) [25]. Longitudinal analysis was performed using a Kohonen-maps based classifier as described in [26], using the CDS statistical test [27].

Reference

Catherine Kamtchueng, Marie-Ã‰ve StÃ©benne, AurÃ©lie Delannoy, Emmanuelle Wilhelm,
HÃ©lÃ¨ne LÃ©ger, Arndt G Benecke and Brendan Bell (2014)  Identification of cis-acting RNA elements that control the expression of the proapoptotic transcription factor TAF6delta.