Abstract
Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.
Highlights
The ribosomal RNA genes encode the catalytic and structural RNAs of the ribosome as a single 47S precursor
The 200 Ribosomal RNA gene copies account for 80% of total cellular RNA and determine the cell’s ribosome complement and its capacity to synthesize proteins. These genes directly control cell growth and their misfunction leads both to hypertrophic and developmental disorders. It is still unclear what determines the choice of which rDNA copies are activated and how this relates to the formation of the transcription preinitiation complex
Our study, based on the conditional deletion of TAF1B, a subunit of the TATA-box Binding Protein (TBP) complex Selectivity Factor 1 (SL1), reveals how the DNA sequence non-specific HMGbox factor UBTF plays a sequence-specific role in SL1 recruitment and preinitiation complex formation
Summary
The ribosomal RNA (rRNA) genes encode the catalytic and structural RNAs of the ribosome as a single 47S precursor. Transcription of the several hundred tandemly repeated and essentially identical [7] rRNA genes, the rDNA, is undertaken exclusively by RNA Polymerase I (RPI, Pol, POLR1) and a set of basal transcription factors dedicated to this task. This strict correspondence of gene and polymerase has resulted in the rapid coevolution of rDNA promoters with basal factors, leading to a high degree of species specificity of the RPI transcription machinery [8,9,10]. What directs the RPI transcription machinery exclusively to the rDNA and how it is recruited to both the major 47S pre-rRNA promoter and enhancer element despite these having little or no DNA sequence commonality are still not understood. We show that this UBTF function is compromised by an E210K mutation recently linked to a recurrent human pediatric neuroregression syndrome [6,12,13,14]
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