FRCPath – December 2008

1. What Mechanisms are used to control eukaryotic gene expression after the production of a fully processed mRNA? FRCPath – December 2008

2. Control of gene expression • Definition - Gene expression is a highly specific process in which information encoded in a gene is translated into a protein or RNA. It can be controlled at various stages depending on the needs and requirements of the cell, tissue or developmental timing. • Stages of control: • Pre transcription • epigenetic gene silencing • chromatin modification • Transcription • Interaction of transcription factors • competition between gene silencers and enhancers • mRNA splicing • Post transcription / Pre translational • mRNA stability • mRNA transport • mRNA degradation • control of translation • Post translational • correct protein folding

3. There is much overlap between the processes on gene regulation at the post translational stage: • mRNA stability • Pre-translational control • RNA-binding proteins • micro RNAs (miRNA) and small interfering RNA (siRNA) • Therapeutics

4. mRNA stability • Gene expression is controlled not only by the rate of mRNA synthesis but also by its rate of degradation. • Several structural characters of mRNA aid stability: • 5’7Methylguanosine cap • The cap provides protection from degradation from endonucleases. Methylation of the cap also helps to promote expression. • Poly-A tail • Poly-A binding proteins bind to the tail and prevent degradation by endonucleases. • Shortening of the poly A tail can induce degradation. This process can be accelerated by binding of miRNAs • AU-rich elements (or ARE) located in the 3’UTR (particularly of proto oncogenes and some transcription factors that need rapid degradation) have been identified. Degradation or stability is mediated via RNA binding proteins. • Incorrectly spliced mRNA, particularly those retaining introns, are degraded (via the NMD pathway?)

5. Pre-translation control • There are a few regulatory aspects of the mRNA that determine correct translation into the protein product. • m7G cap • The presence of a m7G Cap promotes translation • Context of the initiation codon i.e. Kozak sequence • Kozak sequence is accAUGG (purine -3 and G +4 important) • Leaky scanning results if AUG is in a suboptimal context – ribosome skips to the next AUG. • 5’UTR • The presence of secondary structures can hinder ribosome binding depending on the length of the 5’UTR and proximity to the 5’cap structure. • Short UTR can also hinder binding – diameter of eukaryotic ribosome is approx 30nt. • Regulatory sequences in the 5’UTR and 3’UTR (see next slide)

6. RNA-binding proteins (5’UTR) • RNA-binding proteins generally have binding sites at 5’UTR or 3’UTR. • RNA-binding proteins can function in a number of different ways • Binding may alter the RNA structure to either facilitate or hinder interactions with trans-acting factors • They may provide localization or targetting signals for the transport of RNA molecules to distinct intracellular locations. • Example = Ferritin is the intracellular iron storage molecule and contains an Iron Response Element (IRE) in its 5’UTR At low cellular iron levels (i.e. no iron for storage) the Iron Response Protein (IRP) binds the IRE and prevents translation. During high cellular iron levels the IRP does not bind the IRE and translation in allowed. . Day & Tuite 1998 J Endocrinology 157 361-371

7. RNA-binding proteins (3’UTR) • At the 3’ end of the mRNA RNA-binding proteins have been shown to regulate endonucleolytic cleavage sites. • Example = Transferrin receptor (TfR) is involved in iron homeostasis and is important for transport of iron into the cell. It is regulated by iron levels in the cell. The transferrin receptor mRNA contains a number of Iron Response Elements (which form a hairpin secondary structure) within the mRNA in its 3’UTR. In between these IREs are endonuclease cleavage sites. At high cellular iron levels within the cell (i.e. no need for more transport of iron into the cell. Endonucleases are allowed to access the cleavage sites and render the mRNA inactive. At low iron levels (i.e. when more iron is needed within the cell) Iron Response Proteins (IRP) bind to Iron Response Elements (IRE) in the 3’UTR and render the cleavage sites inaccessible to endonucleases and therefore allowing translation. l Day & Tuite 1998 J Endocrinology 157 361-371

8. RNA-binding proteins associated with disease • RNA binding proteins are thought to contribute to the disease pathology of some of the poly glutamine diseases. RBP bind the poly Q tracts in normal individuals, therefore in poly expansion many more RBP bind and aid aggregate formation. • FMRP is a selective RNA-binding protein that regulates the translation of a subset of mRNAs at synapses. In fragile X syndrome a trinucleotide repeat expansion in the promoter region become hypermethylated and inactivates the FMR1 gene, which leads to the absence of the fragile X mental retardation protein FMRP. In the absence of FMRP, excess and dysregulated mRNA translation leads to altered synaptic function and loss of protein synthesis.

9. Micro RNA (miRNA) • Micro RNAs are a family of non translated RNAs, 21-25nt in length, that negatively regulate gene expression at the post transcriptional level by 1 of two mechanisms: • Translational repression • mRNA cleavage (a mechanism shared with small interfering RNAs (siRNA)) • siRNA and miRNA are have many similarities: • Molecular characteristics • Share a common RN’ase III processing enzyme called DICER • Both associate with effector complexes called ‘RNA induced silencing complexes’ (RISC) • The main differences between miRNA & siRNA are: • Where miRNA are produced from specific precursor encoded in the genome, siRNA are sampled randomly from long dsRNA that are either introduced exogenously or produced endogenously by bi-directional transcription that anneals to form dsRNA. • miRNAs bind to 3’UTR regions by imperfect complimentarity at multiple sites whereas siRNA form duplexes with targets at one site and direct cleavage of target mRNA at the site of complimentarity.

10. miRNA – Translational repression • Nascent miRNA transcripts (pri-miRNA) are processed into 70nt precursors. They are cleaved by DROSHA (now called pre-miRNA) • Pre-miRNA exported from the nucleus where a second round of processing is undertaken – DICER cleaves into 21-25nt fragments • One strand of the duplex is incorporated into the RNA induced silencing complex (RISC) which then binds the target mRNA with partial complimentarity • Translation is inhibited. He & Hannon 2004 NRG5 522

11. miRNA & siRNA – mRNA cleavage • This mechanism of gene regulation is stimulated by the presence of dsRNA ~500bp long. • The dsRNA is processed into small dsRNA molecules of 21-25nt (siRNA). • As previous, one strand of the duplex is incorporated into the RISC and the RISC binds complimentary mRNA and directs cleavage. • The cleavage process is known as ‘post transcriptional gene silencing’ (PTGS) • It is thought that this was an evolutionary mechanism for defence against virus infection. He & Hannon 2004 NRG5 522

12. Micro RNAs associated with disease (e.g. Cancer) • miRNAs play important roles in homeostatic processes such as development, cell proliferation and cell death. Recently the dysregulation of miRNAs has been linked to cancer initiation and progression, indicating that miRNAs may play roles as tumour suppressor genes or oncogenes: • Array based micro RNA expression profiling had identified a series of upregulated and down regulated miRNA in medulloblastoma cells, when compared to normal adult and fetal cerebellar tissue. .

13. Control of gene expression – potential for therapeutics • RNAi – can be used to inhibit translation of specific proteins. Potential targets include virus’ and oncogenes. No clinical trials in humans as yet. • Ribozymes (RNAs with enzymatic functions). They can cleave targetted RNA in a sequence specific manner. After it is released from the RNA it can find and bind a new target. Potential as therapy for viruses e.g. Hepatitis B

14. References • Day & Tuite 1998 Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview. J Endocrinol 157 361-371 • He & Hannon 2004 MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genetics 5 522-531 • Harfe 2005 MicroRNAs in vertebrate development. Curr Opin Genet & Devel 14 410-415 • Siomi & Dreyfuss 1997 RNA-binding proteins as regulators of gene expression. Curr Opin Genet & Devel 7 345-353 • www.wikipedia.co.uk • Doi et al 2008 JCB 283(10) 6489-500 • Bassell & Warren 2008 60(2) 201-16