Saturday, 22 December 2012

RNAP I, III, IV and V transcription systems

RNA polymerase I directed rDNA transcription, life and works by Russell and Zomerdjik
Figure 3. The RNA polymerase I (pol I) transcription cycle: pre-initiation complex formation (PIC), transcription initiation, promoter escape and clearance, elongation, termination and reinitiation. (1)De novo PIC formation involves the selective binding of selectivity factor 1 (SL1) to the rDNA promoter, the incorporation of activator upstream binding protein (UBF) and (2) the recruitment of Pol Iβ by SL1. (3) Pol I initiates transcription upon promoter opening and, following promoter escape (3), pol I is converted into a processive enzyme (pol Iε), which elongates the nascent rRNA (4)(5)Transcription by pol I terminates at the 3′end of the gene at specific sequences bound by termination factor TTF-I and transcript-release factor PTRF, with the concomitant release of pol I and the nascent rRNA. (6) SL1 and UBF remain promoter-bound following promoter clearance by pol I, and form a reinitiation scaffold onto which a pol Iβ complex, perhaps generated from recycled pol I and hRRN3, is recruited, and the resultant productive PIC can initiate another cycle of transcription.

O'Sullivan 2002 Mol Cell Bio 2002: UBF binding is not restricted to regulatory sequences in  vertebrate rDNA repeat
FIG. 4.
UBF binds extensively over the Xenopus rDNA repeat. (A) The structure of the Xenopus rDNA repeat. Solid black boxes indicate 18S, 5.8S, and 28S rRNA coding sequences. Open boxes represent 5′ and 3′ external transcribed spacers and internal transcribed spacers. Clusters of vertical bars represent blocks of enhancer elements. The scale bar below represents the length of the repeat in kilobases. The position and size of subclones 1 to 10 of the Xenopus rDNA repeat are shown below. (B) Duplicate arrays of subclones 1 to 10 (slots 1 to 10) and vector controls (slots C) were arrayed and probed with radiolabeled cloned rDNA repeat (top) and DNA extracted from both preimmune (middle) and α-xUBF (bottom) ChIP assays. Relative UBF loading values are shown below.



FIG. 8.
RNA Pol I and SL1 show a restricted distribution on the human rDNA repeat. (A) PCR was performed with DNA extracted from preimmune, α-UBF, and α-Pol I ChIP assays with primer pairs from across the human rDNA repeat. The locations of the primer pairs and gels of the resulting PCRs are shown in the appropriate position below a diagram of the human rDNA repeat. The source of DNA used in each PCR is shown below the gel. (B) PCR was performed with DNA extracted from α-TafI110 and α-TafI48 ChIP assays with primer pairs from across the human rDNA repeat. The identity of the primer pairs is shown above the appropriate gel lanes, and the identity of the antibody used in ChIP is shown alongside.
RNA Pol I selectively cross-links to transcribed sequences in the human rDNA repeat.Whereas UBF binds across the entire rDNA repeat in both Xenopus and human cells, one would expect that other proteins related to ribosomal gene transcription would show a more restricted distribution. In particular, α-Pol I antibodies should only immunoprecipitate transcribed sequences within the rDNA repeat. To test this, we performed PCRs with primer pairs targeted to both transcribed and nontranscribed sequences with DNA extracted from α-Pol I ChIP assays (Fig. 8). The α-Pol I antibody used in this experiment (S18) has been extensively characterized and demonstrated to bind engaged Pol I

Significant amounts of PCR product are recovered from both α-UBF and α-Pol I ChIP assays with primer pairs H1, H4, H8, and H13 that fall within the 47S coding sequence. Product is observed from α-UBF but not α-Pol I ChIP assays with primer pairs H23/27 and H42 that fall within the IGS (Fig. 8A). Thus, we can confirm that this assay shows the expected and restricted distribution of RNA Pol I on the human rDNA repeat. Note that the absence of Pol I over sequences targeted by the H42 primer pair (1 kb upstream of the transcriptional start) confirms the resolution of this technique.
SL1 binding is restricted to promoter sequences.SL1 is a component of the stable transcription complex that forms at the promoter. Consequently, one would expect that SL1 should show a more-restricted distribution than either UBF or Pol I. ChIP experiments were performed with antibodies that recognize TafII48 and TafI110 components of SL1. PCR was performed with DNA recovered from each ChIP with primer pairs from across the rDNA repeat (Fig. 8B). Promoter sequences and sequences up to 1 kb downstream of the transcription start site are present in α-TafI48 and TafI110 ChIP experiments. As expected, sequences elsewhere in the transcribed region (H4, H8, and H13) and in the IGS (H18 and H23/27) are absent.


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.
FIG. 3.
Repression of RNA Pol I transcription in vitro by recombinant p53. (A) HeLa nuclear extracts were preincubated for 20 min at 30°C with either GST (lanes 1, 4, and 5, 2, 2, and 6 pmol, respectively, as judged by silver-stained gels), an increasing amount of GST-p53 (lanes 2 and 3 and lanes 6 and 7, 2 and 6 pmol, respectively), or an increasing amount of GST-p53(his175) (lanes 8 and 9, 2 and 6 pmol, respectively) before the addition of transcription template prHu3 (5 ng/reaction mixture) and nucleotides to initiate transcription. GST, GST-p53, and GST-p53(his175) were expressed inE. coli and were affinity purified using glutathione-Sepharose beads. 
As shown in Fig. 3A, the Pol I transcription was unaffected by the addition of the mutant form of p53 (lanes 8 and 9). Thus, our data indicate that p53 acts directly on the Pol I transcription machinery to inhibit rRNA synthesis.


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. 

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