Similar colocalization was observed in situ, in brain sections from old mice. The presence of TERT in RNA-rich organelles and its affinity for G-quadruplex structures, which are also present in RNA, motivated us to investigate Olaparib whether cytoplasmic TERT is in fact associated with RNA/ribosomes. To analyze this possibility, cytoplasmic extracts from mouse brain were fractionated on a sucrose gradient, and the cosedimentation of TERT with polysomes/messenger-ribonucleoparticles was determined by Western blotting. As shown in Fig. 1e, the largest pool of TERT co-sediments mostly with ribosome/monosomes and the 80S initiation complex, partially with RNA bound to two and tree ribosomes and only a small amount of the protein was detected in mRNPs and polysome fractions. To ascertain whether TERT was indeed binding ribosomes, polypepetide elongation was interrupted with puromycin. Fig. 1f shows that this treatment induces a shift of TERT towards the lighter fractions of the gradient, strengthening the notion of a TERT/ribosome association. When subjected to environmental stress, cells respond by suspending overall protein synthesis, resulting in the disassembly of polysomes and the stalling of initiation complexes, which become recruited to cytoplasmic foci known as stress Tofacitinib granules. Because cytoplasmic TERT favors neuronal survival and co-sediments mostly with monosomes and the initiation translational complex, we tested whether TERT is part of SGs. We immunoprecipitated TERT from extracts of hippocampal neurons in vitro and determined its possible association with the RNA binding protein TIA1, a well-known marker of SGs. TIA1 and TERT coprecipitated in the cytosolic fraction from mature hippocampal neurons in culture. A similar result was observed when the immunoprecipitation was performed in brain cytoplasmic extracts from old mice, together suggesting that TIA1 and TERT are in a complex. Because arsenite increases the number of SGs, we treated fully differentiated hippocampal neurons in culture with 0.5 mM arsenite for 60 min. Western blot analysis shows that arsenite treatment does not produce any significant increase in TERTTIA1 complexes, suggesting that at this stage of neuronal differentiation, the number of complexes is at the upper limit. Moreover, RNase treatment does not affect the binding of TERT to TIA1, demonstrating that the association is RNA independent. In further support that TERT is part of SGs, coimmunoprecipitation experiments revealed the presence of PeIF2a, another specific SGs marker.