Abstract
Altered expression of NEAT1, the architectural long non-coding RNA (lncRNA) of nuclear paraspeckles, has been reported during tumorigenesis, as well as under various cellular stress conditions. Here we report that the depletion of NEAT1 lncRNA alleviates nucleolar stress during RNAP I inhibition through releasing sequestered P54nrb and PSF to facilitate the IRES-dependent translation of c-Myc. RNAP I inhibitor CX5461 disrupts the SL1-rDNA interaction and induces nucleolar disruption, demonstrated by the accumulation of fibrillarin-containing nucleoplasmic foci and nucleolar clearance of ribosomal proteins in HeLa cells. Antisense oligonucleotide-mediated depletion of NEAT1 lncRNA significantly attenuated the RNAP I inhibition and its related nucleolar disruption. Interestingly, induction in the levels of c-Myc protein was observed in NEAT1-depeleted cells under RNAP I inhibition. NEAT1-associated paraspeckle proteins P54nrb and PSF have been reported as positive regulators of c-Myc translation through interaction with c-Myc IRES. Indeed, an increased association of P54nrb and PSF with c-Myc mRNA was observed in NEAT1-depleted cells. Moreover, apoptosis was observed in HeLa cells depleted of P54nrb and PSF, further confirming the positive involvement of P54nrb and PSF in cell proliferation. Together, our results suggest that NEAT1 depletion rescues CX5461-induced nucleolar stress through facilitating c-Myc translation by relocating P54nrb/PSF from nuclear paraspeckles to c-Myc mRNAs.
Highlights
Paraspeckles are mammalian-specific RNA-protein structures with more than forty identified protein components [1]
To better understand the function of NEAT1 long non-coding RNA (lncRNA) during cellular stress related to transcriptional inhibition of RNAP I, we depleted NEAT1 by antisense oligonucleotides (ASOs) designed to target the 5’-region shared between NEAT1 isoforms (NEAT1_1 and NEAT1_2) through a RNase H1-dependent mechanism (Fig 1A)
To test the hypothesis that the depletion of NEAT1 lncRNA can facilitate internal ribosome entry segment (IRES) translation of c-Myc by redistributing P54nrb and PSF from nuclear paraspeckles onto the c-Myc mRNA, we examined the association of P54nrb or PSF with c-Myc mRNA by RNA binding protein immunoprecipitation (RNA-IP) (Fig 5I and 5J)
Summary
Paraspeckles are mammalian-specific RNA-protein structures with more than forty identified protein components [1]. Paraspeckles assemble hierarchically through the recruitment of protein components to the central scaffolding long non-coding RNA, NEAT1. NEAT1 RNA is indispensable to the maintenance of paraspeckle integrity. Complete disappearance of paraspeckles was reported in NEAT1-/- MEF, while the exogenous expression of mouse NEAT1_2 in NEAT1-/- MEF rescued paraspeckle formation [2]. Paraspeckles are absent in embryonic stem cells due to the lack of NEAT1 expression, but are formed immediately upon differentiation following the expression of NEAT1 [3].
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