O'Sullivan 2002 Mol Cell Bio 2002: UBF binding is not restricted to regulatory sequences in vertebrate rDNA repeat
Repression of RNAPI transcription by teh tumour suppressor p53 by Zhai and Comai
The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. p53 may repress RNAPI tranctipion.
They examined molecular mechanism of Pol I transcription inhibition by p53.
We show that wild-type, but not mutant, p53 can repress Pol I transcription from a human rRNA gene promoter in cotransfection assays. Furthermore, we show that recombinant p53 inhibits rRNA transcription in a cell-free transcription system. In agreement with these results, p53-null epithelial cells display an increased Pol I transcriptional activity compared to that of epithelial cells that express p53. However, both cell lines display comparable Pol I factor protein levels.
Our biochemical analysis shows that p53 prevents the interaction between SL1 and UBF. Protein-protein interaction assays indicate that p53 binds to SL1, and this interaction is mostly mediated by direct contacts with TATA-binding protein and TAFI110. Moreover, template commitment assays show that while the formation of a UBF-SL1 complex can partially relieve the inhibition of transcription, only the assembly of a UBF-SL1-Pol I initiation complex on the rDNA promoter confers substantial protection against p53 inhibition. In summary, our results suggest that p53 represses RNA Pol I transcription by directly interfering with the assembly of a productive transcriptional machinery on the rRNA promoter.
The Myc trilogy: lord of RNA polymerases by Oskarsson and Trumpp
Oncoprotein Myc enhances rRNA synthesis by RNAPI. It also controls RNAPII and II regulated gene transcription. Myc may promote generation of crucial components of a functional ribosome.
Human cancers carry mutations that cause inactivation of tumour suppressor genes and activation of oncogenes. Mnay of these gnes control cell cycle. Thus it was thought that deregulation of cell division is the principal mechanism that drives tumour progression.
However it is discovered that increased cell division requires boost in growth. Onco and tumour suppresor proteins are crucial for growth control and protein synthesis.
The oncoprotein Myc controls rRNA synthesis by RNA Pol I, a rate limiting step for cell growth.
Proliferation cannot occur without enough cell growth. Ribosomes are factories for protein synthesis. They comprise 4 different rRNAs.
In nucleoli, transcriptionally active rRNA genes cluster. RNAPI transcribes a 45s precursor rRNA. It is processed into smaller rRNAs that form scffold and catayltic centre of ribosome. In nucleoplasm, ribosomal proteins and 5s rRNAs are synthesised by RNAP II and III. Before being transported to nucleoli where pre-ribosome assembly occurs.
myc encodes a TF that activates or represses 2 sets of target gemes.
Genomic and proteomic approaches showed differential expression of RNAP II traget genes encoding ribosomal and nucleolar proteins in cells overexpressing Myc.
C-Myc activates RNAPIII transciprtion by interacting with TFIIIB. C-myc is suggested to influence 45s rRNA processing.
c-Myc influences rNRA transcription indirectly by controlling expression of UBF essential for RNAPI mediated transcription.
ARabi and Randori showed that c-Myc directly regulates RNAPI transcription. Using gain and loss of function, they showwed that c-Myc controls rRNA synthesis in mammalian cells. The effects are still observed in presence of RNAPII inhibitor, this suggests that c-Myc directly activates Pol I transcription. Confirtmed by ChIP experiments. Showed presence of c-Myc at E box elements in rDNA promoter.
In mammals, RNAPI assembly of PIC inovlves SL1 which comrpsies TBP and 3 TAFs. Interaction of SL1 and TAF-1A recruits pol to rDNA promoter.
Grandori showed that c-Myc associates with TBP and TAF of SL1 complex. TBP association with promoter increases with high levels of c-Myc. It decreases on c-Myc downregulation. c-Myc positively regulates efficiency of Pol I recruitment to target promoters. Enhanced c-Myc association with rDNA promoter correlates with increased acetylation of H3 and H4. As for Pol II- transcribecd genes, c-Myc may recruit HATs to rDNA promoter to regulate Pol I transcription.
The Odd Pols are even when it comes to ocntrolling cell function by Hanna and Schultz
Cancer cells produce ribosomes at higher rate than normal cells. Perinuclear compartment (PNC) is enriched in a RNAP III transcript required for pre-rRNA processing. PNC prevalence correlates with malignanct and metastatic behaviour of human breast and prostate cells.
Nucleolar localisation of tRNA genes in budding yeast connects RNAPI and RNAP III system modules at level of nuclear organisation. Nucleolar localisation of tRNA genes depends on microtubuels. tRNA genes cluster together. Clustering requires condensin. It is concentrated at tRNA and some RNAPIII-transcribed genes.
Ayoub showed that constitutive activation of RNAPI causes accumulation of 5s rRNA, mRNAs encoding ribosomal proteins and fully assembled ribosomes.
Using 3D DNA-immuno-FISH, distal junctions of Nucleolar organiser regions (NORs) were shown to be located in heterochromatin surrounding nucleoli.
Non-coding RNA production by RNA polymerase II is implicated in cancer by Marshall and White
RNAPIII is largest RNAP. It has 17 subunits.
Increased Pol III output in cancers
3 general mechanisms that cause pol iii output in cancers.
1) tumour suppressors erelease from repression
2) activation by oncogene products
3) increased expression of poll III-associated TFs.
Release from tumour suppressors
PTEN, p53 and RB are tumour suppressors whose activities are compromised in some cancers.
PTEN inhibits Poll III transcription. may counteract signalling through PI3K pathway.
PolIII-dependent expression of tRNA and 7SL RNA increases after depletion of PTEN or activation of PI3 signalling.
Correlate with changes in target gene occupancy by Pol III and its associated TF, TFIIIB. TFIIIB is required to recruit pol III to any of its templates. Its dissocation may explain decrease in pol iii occupancy and transcription in response to PTEN.
TFIIIB is a target for p53, RB and RB-like proteins. They bind it directly and prevent it recruiting Pol iii to promoters.
Activation by oncogene products
TFIIIB is reulated by ncogenic proteins that stimulate its activity. Push TFIIIb into active state.
Many oncogenic products subvert restraints imposed by RB and p53, affecting TFIIIB indirectly. Eg E6 and E7 oncoproteins of HPV can stimulate pol iii transcription by inactivating p53 and RB family.
Others eg Ras, Raf, PI3K and Akt alter phosphorylation state of Pol iii machinery.
Transforming proteins act directly on TFIIIB to stimulate its activity eg MYC.
MYC induction increases target occupancy by TFIIIB and Pol III. This correlates with localised acetylation of histone H3, which is associated with active transcription. RNAi was usd to partially deplete cells of TFIIIB. Cells maintained normal levels of PolIII prodcuts but no increase in repsonse to MYC. Affected ability of MYC to drive colony formation in sot agar and tumour growth in mice.
Overexpression of Pol III transcription factors
High levels of Pol III products in smoe tumours may be cause by overexpression of transcription machinery. Levels of TFIIIC levels can be increased in culture after infection or transformation by DNA tumour viruses eg adenovirus, simian virus 40 and Epstein barr virus. TFIIIC overexpression is high in human ovarian and naopharyngeal carcinomas.
Overexpression of Pol iii-specific TFs is advantageous for cancer.
Consequences of raising pol iii transcription
Clone cells with transfected copies of cDNA encoding BRF1, a subunit of TFIIIB. BRF1 recruits Pol iii to templates. Increased BRF1 expresssion was found in some cervical carcinomas.
Inducing BRF1 raised pol iii occupancy at tRNA and 5s rRNA genes and increased expression of their transcripts. Increased proliferation Cell numbers with BRF1 induction were higher than uninduced cells or control cells with empty vector. MEF and Chinese hamster ovary cells underwent oncogenic transformation.
Phenotype produced by BRF1 overexpression acn be recapitulated by overexpressing one of its pol iii- transcribed targets, a gene encoding tRNAi Met. (tRNA required for initiating polypeptide synthesis). Immotalised MEFs were transfected with extra copies of this gene. Increased levels of tRNAimet.
Increased proliferation, focus formation, anchorage independence and ability to form tumours when injected into mice - markers of transformation. BRF1 induction effects may be caused by increased ni tRNAimet expression.
Rate of total protein synthesis increased in response to tRNAi or BRF1. Consistent with tRNAi acting at rate limiting step in translation, pp chain initiation.
Levels of cyclin D1 and MYC increase preferentially in response to tRNAimet and BRF1 overexpression. Levels of their corresponding mRNAs do not show this change.
A chance for therapeutic intervention
Restricting Pol iii transcription may be a therapeutic strategy. RNAi-mediated partial depletion of BRF1 can inhibit focus formation by colon cancer cells in culture. Reduce tumour growth when MYC-transformed fibroblasts are injected into mice. Would a drug that produces global reduction of pol iii output be toxic.
The role of UBF in regulating structure and dynamics of transcriptionally active rDNA chromatin by sAnij and Hannan
3 distinct rDNA chromatin states: transcriptionally competent, traenscriptionallyactive and inactive rDNA. Active rDNA genes in yeast have open chromatin structure, associated with nascent RNA and accessible to psoralen. Silent genes are inaccessibly to psoralen and associated with regularly spaced nucleosomes.
Psoralen accessible euchromatic state of r-chromatin is maintained in mitotic cells. rRNA genes are transcriptionally silent. This suggest that euchromatic r-chromatin can be actively transcribed or transcriptionally competent bu unproeudctive. Not all euchromatic rRNA genes are transcived.
UBF maintains euchromatic state of rDNA chromatin. UBF maintains undercondensed r-chromatin of active NORs.