Lecture 4 Regulation of Initiation: mrna-binding

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1 Institut für Biochemie und Molekulare Medizin Lecture 4 Regulation of Initiation: mrna-binding Michael Altmann FS 2010

2 Regulation of mrna binding to ribosomes mrna structure Cap accessibility RNA secondary structure Upstream open reading frames(uorf s) AUG context (positions -3 and +4) protein-mrna interactions 5 UTR (e.g iron homeostasis) 3 UTR (e.g. ceruloplasmin mrna) mrna localization (e.g. β-actin mrna) small RNA s translation initiation mrna half-life eif modification modulation of eif4g-eif4e interaction degradation/processing of eif4g

3 Reference: MW Hentze et al. Cell 117: , Iron metabolism

4 Regulation of iron metabolism Iron Responsive Element-B / IR A cytoplasmatic aconitase (additional to the one located in the mitochondria), converts citrate to isocitrate. When iron concentration is high in the cell, an ironsulphur cube is formed that determines structure and enzymatic activity of the aconitase. When iron levels are low, the iron-sulphur cube disintegrates, the aconitase structure changes and adquires the property to bind to the IRE (iron responsive element) of regulated mrnas.

5 Iron metabolism

6 Steric hinderance prevents recruitment of 40S-ribosomes

7 Role of IR Reference: MW Hentze et al. Cell 117: , 2004

8 Reference: F Gebauer & MW Hentze Nat.Rev.Mol.Cell.Biol. 5: , Role of the 3 UTR

9 Reference: J Coller & R arker Cell 122: , body formation

10 β-actin mrna transport and translation β-actin mrna β-actin Reference: S Hüttelmaier et al. Nature 438: , 2005.

11 Nomenclature of small RNAs (22 nucleotide long) MicroRNAs match unperfectly to mrna leading to translational inhibition (reversible) Small interfering RNAs (sirnas) match perfectly to mrnas which are degraded (irreversible) A small hairpin RNA or short hairpin RNA (shrna) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shrna uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shrna is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shrna hairpin structure is cleaved by the cellular machinery into sirna, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mrnas which match the sirna that is bound to it.shrna is transcribed by RNA polymerase III. shrna production in a mammalian cell can sometimes cause the cell to mount an interferon response as the cell seeks to defend itself from what it perceives as viral attack. This problem is not observed in mirna, which is transcribed by RNA polymerase II (the same polymerase used to transcribe mrna). def. from Wikipedia

12 mirna and translation Reference: F Gebauer & MW Hentze Nat.Rev.Mol.Cell.Biol. 5: , 2004.

13 Regulation of eif4e- Binding roteins Richter & Sonenberg, 2005

14 Regulation of eif4e-b activity receptor A B eif4g I3 I3 I3K KB DK eif4e 4E-B nutrients TO mrna translation Y X mtor p70s6k cap mrna 4E-B does not bind to eif4e 4E-B binds to eif4e rps6 eif4b translation initiation up translation initiation down translation translation translation protein phosphatase Tap42/alpha4 mtor, stimulation;, inhibition;, enhanced;, lowered.

15 Reference: M Holz et al. Cell 123: , eif3-tor interaction

16 eif4g cleavage 1970 s: poliovirus infection leads to shut-off of host mrna translation Inhibition of capped mrna translation but not of polioviral RNA (uncapped) A viral protease cleaves the eif4e-binding domain off eif4g. The remaining eif4g fragment is sufficient to stimulate internal initiation. Certain host mrnas are translated by internal initiation. This may be important in stressed cells, during mitosis and apoptosis.

17 Reference: M Holcik & N Sonenberg Nat.Rev. Mol. Cell. Biol. 6: , eif4g cleavage and apoptosis

18 Seminar 1 - What are the biol. consequences of mrna transport and localized translation? - Inform yourself about CCA adding enzyme (trna)! - How many rrna genes does a cell need to make 10 6 ribosomes in 6 hours? - What are the most prominent differences in the mechanism of initiation between prokaryotes and eukaryotes? - Why should a cell need so many helicases? - What s about translation in mitochondria? - Are there orthologs of eif s? - How are protein-protein interactions measured? - Why are 10-20% of eif2α- sufficient to block translation? - hysiological consequences of translational regulation of transcription factors? - Viruses fight against eif2 kinases! How? - What do you know about the roles of small RNAs in gene expression?