mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons

Abstract

mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon–exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5′-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.

BAX to basics: How the BCL2 gene family controls the death of retinal ganglion cells

“…Additionally, because BAK is already located at the MOM, only BAX has the potential to be retro-translocated. It may be no coincidence, then, that neurons, which only express a functional BAX protein (Jakobson et al, 2012; Uo et al, 2005) (see below), rely principally on the function of BCL-X (Deckwerth et al, 1996; Levin et al, 1997; Mosinger Ogilvie et al, 1998; White et al, 1998). Whether this retro-translocation occurs when BAX is inactive or active, remains to be determined.…”

“…Whether or not N-BAK functions as a BH3-only protein is still in question. Forced expression of N-BAK in tissue culture cells promotes apoptosis (Sun et al, 2003; Uo et al, 2005), but endogenously, N - Bak transcripts are in low abundance, and may be translationally repressed through various mechanisms (Jakobson et al, 2012, 2013). Preliminary studies done by our group indicates that N - Bak / N - BAK transcripts are present in retinal neurons of mice and non-human primates.…”

Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons

“…11 When activated, Bak and Bax generate pores on the mitochondrial outer membrane 12 whereby the mitochondrial proteins (cytochrome c , DIABLO/Smac, EndoG, AIF and so on) are released to the cytosol to advance apoptosis. 13, 14 We have shown 15, 16 that unlike all other studied cells, neurons do not express Bak as its pre-mRNA is entirely spliced into mRNA encoding a putative BH3-only protein, named N-Bak. Thus, the neurons express only Bax, 15, 16, 17, 18, 19 and their mitochondrial apoptosis depends solely on Bax.…”

“…13, 14 We have shown 15, 16 that unlike all other studied cells, neurons do not express Bak as its pre-mRNA is entirely spliced into mRNA encoding a putative BH3-only protein, named N-Bak. Thus, the neurons express only Bax, 15, 16, 17, 18, 19 and their mitochondrial apoptosis depends solely on Bax. 20 In the same studies 15, 16 we demonstrated that, despite the presence of mRNA, N-Bak protein is not detectable in the brain or cultured primary neurons.…”

“…Thus, the neurons express only Bax, 15, 16, 17, 18, 19 and their mitochondrial apoptosis depends solely on Bax. 20 In the same studies 15, 16 we demonstrated that, despite the presence of mRNA, N-Bak protein is not detectable in the brain or cultured primary neurons. We also showed 16 that the absence of N-Bak protein is not caused by rapid degradation of its mRNA or proteasome-mediated degradation of the protein.…”