STAT3 promotes RNA polymerase III-directed transcription by controlling the miR-106a-5p/TP73 axis
Curation statements for this article:-
Curated by eLife
eLife assessment
The author arrive at the convincing conclusion that STAT3 expression promotes TFIIIB assembly through miR-106A-5p-mediated inhibition of TP73 expression, thereby increasing Pol III transcription, which contributes to enhanced cell proliferation. The data are very good and clearly support the proposed model.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Molecular Biology (eLife)
Abstract
Deregulation of Pol III products causes a range of diseases, including neural diseases and cancers. However, the factors and mechanisms that modulate Pol III-directed transcription remain to be found, although massive advances have been achieved. Here, we show that STAT3 positively regulates the activities of Pol III-dependent transcription and cancer cell growth. RNA-seq analysis revealed that STAT3 inhibits the expression of TP73, a member of the p53 family. We found that TP73 is not only required for the regulation of Pol III-directed transcription mediated by STAT3 but also independently suppresses the synthesis of Pol III products. Mechanistically, TP73 can disrupt the assembly of TFIIIB subunits and inhibit their occupancies at Pol III target loci by interacting with TFIIIB subunit TBP. MiR-106a-5p can activate Pol III-directed transcription by targeting the TP73 mRNA 3’ UTR to reduce TP 73 expression. We show that STAT3 activates the expression of miR-106a-5p by binding to the miRNA promoter, indicating that the miR-106a-5p links STAT3 with TP73 to regulate Pol III-directed transcription. Collectively, these findings indicate that STAT3 functions as a positive regulator in Pol III-directed transcription by controlling the miR-106a-5p/TP73 axis.
Article activity feed
-
-
eLife assessment
The author arrive at the convincing conclusion that STAT3 expression promotes TFIIIB assembly through miR-106A-5p-mediated inhibition of TP73 expression, thereby increasing Pol III transcription, which contributes to enhanced cell proliferation. The data are very good and clearly support the proposed model.
-
Reviewer #1 (Public Review):
Zhang et al. have submitted a manuscript demonstrating that STAT3 regulates RNA polymerase III transcription in human tumor cell lines. They present several lines of evidence for this proposal. They show that short hairpin (sh)RNAs that repress STAT3 inhibit Pol III transcription and limit proliferation in HepG2, HuH-7, and 293T cells. Accordingly, overexpression of STAT3 enhances Pol III transcription and increases proliferation in the same cell lines. STAT3-dependent EdU incorporation into synthesized DNA confirmed the proliferation results. The Pol-III transcription inhibitor ML-60218 reversed the positive proliferation effects of STAT3 overexpression. In a mouse xenograft model, overexpression of STAT3 enhanced tumor growth of HepG2 cells, whereas suppression of STAT3 inhibited its growth. Consistent …
Reviewer #1 (Public Review):
Zhang et al. have submitted a manuscript demonstrating that STAT3 regulates RNA polymerase III transcription in human tumor cell lines. They present several lines of evidence for this proposal. They show that short hairpin (sh)RNAs that repress STAT3 inhibit Pol III transcription and limit proliferation in HepG2, HuH-7, and 293T cells. Accordingly, overexpression of STAT3 enhances Pol III transcription and increases proliferation in the same cell lines. STAT3-dependent EdU incorporation into synthesized DNA confirmed the proliferation results. The Pol-III transcription inhibitor ML-60218 reversed the positive proliferation effects of STAT3 overexpression. In a mouse xenograft model, overexpression of STAT3 enhanced tumor growth of HepG2 cells, whereas suppression of STAT3 inhibited its growth. Consistent with these results, overexpression of STAT3 enhanced colony formation of HepG2 cells in soft agar, whereas STAT3 suppression inhibited it. ChIP data suggest that STAT3 shRNAs reduce the presence of TBP, BRF1, TFIIIC subunits, and POLR3A at Pol III genes regulated by gene-internal promoters. However, STAT3 does not bind to 5S, 7SL, U6, and tRNA Met genes. In addition, STAT3 does not affect the expression of various Pol III transcription factors. RNA-seq in STAT3-shRNA-expressing HepG2 cells and in shRNA-expressing control cells revealed upregulation of 356 and downregulation of 590 Pol II-transcribed genes. None of the Pol III transcription factors were affected. Among the genes whose expression was enhanced by silencing STAT3 was TP73. Accordingly, overexpression of STAT3 decreased mRNA expression of TP73. To show that TP73 acts downstream of STAT3, the authors demonstrated that HepG2 cells expressing both STAT3 shRNAs and TP73 shRNAs did not exhibit decreased Pol III transcription or proliferation. Consistent with these results, TP73 shRNAs enhance Pol III transcription in HepG2 cells, and overexpression of endogenous TP73 represses Pol III transcription. This inhibition of TP73 is caused by the disruption of TFIIIB assembly. Consequently, TP73shRNAs increase the presence of Pol III factors at Pol III genes without affecting their expression. However, co-IP with alphaTP73 antibodies detected TBP, TFIIIC2, and TFIIICC3 but not BRF1, and vice versa. Moreover, shTP73 enhanced the co-IP of TBP with antiBRF1 antibodies. To discover molecular mechanisms explaining how TP73 expression is indirectly regulated by STAT3, the authors identified miR-106a-5p as a potential regulator. In agreement with a regulatory role, miR-106a-5p mimics reduce TP73 expression and enhance Pol III transcription. Finally, Zhang et al. show that STAT3 binds to the miR-106a-5p promoter and activates miR-106a-5p promoter transcription in a luciferase assay.
Overall, the data presented in this manuscript is clean and convincing and clearly supports the proposed model.
-
Reviewer #2 (Public Review):
These discoveries are strongly supported by a large amount of clear and convincing data. Thus, the expression of seven distinct pol III-transcribed genes covering all types of promoter is shown to increase in three cell lines when STAT3 is overexpressed and to decrease when endogenous STAT3 is depleted. The proliferation of HepG2 liver cancer cells can be increased by STAT3 overexpression and decreased by STAT3 depletion. Crucially, proliferative induction by STAT3 is dependent on increased pol III activity, as it can be blocked using a pol III-specific inhibitor at a concentration that allows normal levels of pol III activity, but prevents further elevation. Growth of HepG2 xenograft tumours in mice is also slowed significantly when STAT3 is depleted. The effects of STAT3 on pol III output are indirect, …
Reviewer #2 (Public Review):
These discoveries are strongly supported by a large amount of clear and convincing data. Thus, the expression of seven distinct pol III-transcribed genes covering all types of promoter is shown to increase in three cell lines when STAT3 is overexpressed and to decrease when endogenous STAT3 is depleted. The proliferation of HepG2 liver cancer cells can be increased by STAT3 overexpression and decreased by STAT3 depletion. Crucially, proliferative induction by STAT3 is dependent on increased pol III activity, as it can be blocked using a pol III-specific inhibitor at a concentration that allows normal levels of pol III activity, but prevents further elevation. Growth of HepG2 xenograft tumours in mice is also slowed significantly when STAT3 is depleted. The effects of STAT3 on pol III output are indirect, mediated by miR-106a-5p. Thus, the knockdown of miR-106a-5p reverses the drop in pol III product expression following STAT3 depletion; conversely, pol III output is stimulated by a miR-106a-5p mimic. Elevated levels of miR-106a-5p correlate with significantly worse prognosis for patients with liver cancer. A key target for miR-106a-5p is a sequence in the 3'-UTR of the mRNA encoding TP73. Complementarity to this sequence allows miR-106a-5p to deplete the expression of TP73 and this is shown to be crucial for STAT3 to regulate the proliferation of HepG2 cells. Furthermore, TP73 is revealed to be a direct repressor of pol III-mediated transcription, an activity not previously known. TP73 is shown to inhibit the assembly of TFIIIB, the factor that is responsible for recruiting pol III to all of its genetic templates. A clear and convincing causal flow can therefore be traced: STAT3 induces miR-106a-5p, which depletes TP73, thereby removing a brake that limits pol III output and cell proliferation.
-
