Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements

Abstract

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

“…8c). The visible part of the N-terminal extension (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46) overlaps with the position of the RPC6 FeS domain (Extended Data Fig. 8d).…”

“…A better understanding of RPC7 is of biomedical relevance as this subunit occurs in two isoforms: RPC7α (also known as RPC32α and POLR3G) and RPC7β (also known as RPC32β and POLR3GL). RPC7α is enriched in embryonic stem cells and cancer cells 14,[34][35][36] , and its overexpression augments tumour transformation 14 . Depleting RPC7α also suppresses viability of prostate cancer cells 36 .…”

The third (III) road to cell transformation

“…In conclusion, the study by Durrieu-Gaillard and colleagues [5] is a promising entry point into an extremely complicated phenomenon whose molecular elucidation will require further studies, and it represents a general benchmark in approaching an intricate cellular process such as oncogenic transformation. Most interestingly, this study discloses several future possible genomic investigations on the regulation of a nutrient-sensing machinery (such as pol III) that might ultimately enlighten our understanding of how normal cells become tumorigenic and consequently expand the roads of therapeutic interventions.…”

“…Durrieu-Gaillard and colleagues [5] have been taking advantage of a previously published protocol [6] that they modified to implement a step-wise process of cell transformation of human fetal lung fibroblasts (IMR90) with defined genetic elements [5]. By expressing papilloma virus E6 and E7, SV40 small t-antigen, constitutive active RAS (RAS-G12V) and the catalytic subunit of the telomerase (TERT) they created 16 different cell lines which represent transformation intermediates of which 6 were chosen by the study as major representatives of the transformation process steps [5]. By employing transcript and protein level measurements the authors investigate the concentration changes for both components of the pol III machinery and selected pol III transcripts across the different steps of cell transformation.…”

“…By employing transcript and protein level measurements the authors investigate the concentration changes for both components of the pol III machinery and selected pol III transcripts across the different steps of cell transformation. They report that albeit during cell transformation cellular levels of the major pol III transcripts did not change dramatically, the levels of several subunits of the pol III enzyme and its cognate transcription factors (namely TFIIIB, TFIIIC and PTF/SNAPc) significantly increased over the transformation process [5]. Therefore, the study for the first time provides an in-depth view on quantitative changes of the pol III machinery during the transformation process and identify potential targets for more precise cancer treatment in order to diminish the oncogenic potential of tumorigenic cells.…”