Translation and translational control in eukaryotes I

Transkript

1 Translation and translational control in eukaryotes I Winterterm 2017/2018 Ernst Müllner, MFPL Department of Medical Biochemistry ernst.muellner@meduniwien.ac.at Helmut Dolznig Institute of Medical Genetics helmut.dolznig@meduniwien.ac.at

2 TransCon I 2017 / 2018 (Stand ) 'Structure' * Bedeutung der TransCon als regulatorisches Prinzip * Die Translations-Maschinerie (Erinnererung und darüber hinaus) * Signaltransduktion und Translation (H. Dolznig) * regulatorische Elemente in den 5' UTRs (TOPs, 5'uORFs, ) * IRESs in viralen und zellulären Systemen / Apoptose TransCon II 2018 (Stand ) 'Function' * Onkogenese durch ribosomale Proteine, mirs, Initiationsfaktoren Dolznig * spezifisch RNA-bindende und regulatorisch wirksame Proteine * mrna Stabilität (NMD, ARE-Elemente, IRE/IRP System) * mrna Export aus dem Zellkern, pioneer round of translation * zelluläre Verteilung von mrnas in der Entwicklung und in differenzierten Zellen * TransCon und Stress (heat shock, amino acid starvation, frühe Apoptose, PERK TransCon I und II sind voneinander unabhängig, I vor II macht natürlich Sinn. *Kein* Thema small and large (nuclear) RNAs (lncrnas, mir, shrna, snornas etc), splicing, Viren (nur punktuell), Bakterien, Pflanzen und unizelluläre Eukaryoten ^-^

3 Why Translational Control? Ideology of lectures

4 Why study translational control? Obvious to cure cancer like everybody else REVIEW Targeting the translational machinery as a novel treatment strategy for hematologic malignancies Patrick R. Hagner, Abraham Schneider, and Ronald B. Gartenhaus Blood 115(11): doi: /blood

5 Why study translational control? Asslaber et al. Treatment free survival of B-CLL* patients with or without alterations in the p53 axis (A) p53 del/mut (B) p53- attenuated (p53 del/mut, SNP309 G/G, del11q) (C) low or high mir-34a expression and (D) correlation of mir-34a expression with lymphocyte doubling time. Similar figure in other ref but not so nice BOTH mir function as tumor suppressors connection mir TransCon onco-mirs in tumorigenesis vs. mirs as tumor suppressors * B-CLL = B-cell chronic lymphatic leukemia (Non- Hodgkin B cell lymphoma)

6 Why study translational control? Dolznig TransCon II Müllner TransCon I + II

7 Why study translational control? Relationships between mrna and protein abundances, as observed in large-scale proteome- and transcriptomeprofiling experiments. mrna transcript abundances only partially correlate with protein abundances, typically explaining approximately one- to two-thirds of the variance in steady-state protein levels, depending on the organism. This trend is evident in data from NIH3T3 mouse fibroblast cells. Vogel & Marcotte, Nat Rev Genet 13(4),

8 Weak correlation between mrna and protein levels I transcripts from various S. cerevisiae genes Apparently, the transcriptome does not faithfully represent the proteome. This as a serious problem e.g. in expression profiling since the proteome and not the transcriptome determines cell phenotype! TIBS 26,

9 Weak correlation between mrna and protein levels I transcripts from various S. cerevisiae genes Figure legend: The correlation between the expression levels of 136 Saccharomyces cerevisiae mrnas and their corresponding proteins is depicted as protein / mrna ratio (arbitrary units) against a running index. Numbers of transcripts per cell for each mrna were deduced from SAGE (serial analysis of gene expression) data; protein levels were determined after separation of samples in 2D gels, followed by quantification and identification of the spots. The data reveal a poor correlation between mrna and protein abundance (r = 0.57). Indeed, 72% of the genes analysed fall outside a confidence interval, being twofold higher or lower than the average ratio. Protein abundance can vary more than 20-fold for a given expression level of a particular mrna. Conversely, for a given amount of protein, the expression of the corresponding transcript can differ by up to 30-fold. Gygi, S.P. et al. (1999) Correlation between protein and mrna abundance in yeast Mol. Cell. Biol. 19, Futcher, B. et al. (1999) A sampling of the yeast proteome Mol. Cell. Biol. 19, TIBS 26,

10 Weak correlation between mrna and protein levels II same mammalian mrna in diverse cell types and tissues raw data from: Anderson, N.L. and Anderson, N.G. (1998) Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis 19, Tew, K.D. et al. (1996) Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program. Mol. Pharmacol. 50, Legend: The correlation between mrna and protein abundance for glutathione-s-transferase in 57 human cell lines derived from nine different tissues represented as in (a). Here, the correlation is even lower (r = 0.43). There is a greater than 40-fold variation with respect to the average protein:mrna ratio (aqua line). Apparently, protein levels cannot be reliably predicted from mrna abundance. TIBS 26,

11 Weak correlation between mrna and protein abundance due to widely different ribosome-loading of individual mrnas as determined in POLYSOME GRADIENTS linear sucrose density gradients, 15-40% saturation Assembled polysomes sediment fastest, followed by monosomes, ribosomal subunits and free mrnps. Inherent problem in expression profiling TIBS 26,

12 LEGEND to previous figure Different mrnas are translated with widely different efficiencies Cytoplasmic extracts from primary human activated T cells were fractionated in sucrose gradients, and the RNA from each fraction analysed by northern blotting. The ribosomal RNA distribution profile (28S, 18S and 5S rrna; indicated by arrowheads in the top panel) enables the distinction of fractions comprising mrnps and mrnas engaged in pre-translational complexes (fractions 1 10; lightblue area, designated as free ), and those containing the polysome-bound mrnas (fractions 11 20; dark-blue area, labelled bound ). Hybridization with probes specific for the mrnas encoding p38mapk (red), GADD153 (green) and NT-4 (purple) revealed extensive variations in mrna distribution in sucrose gradients. In the corresponding distribution profiles (bottom), intensities for each fraction are plotted as a percentage of the total signal on the filter for a given mrna to facilitate comparison. The differential distribution of these mrnas implies different translation efficiencies. This phenomenon appears to be much more common than previously anticipated, and is not restricted to particular transcripts, cell types or species. Abbreviations: GADD153, growth arrest and DNA damage; MAPK, mitogen-activated protein kinase; NT-4, neurotrophin 4. TIBS 26,

13 Conventional mrna expression profiling only detects transcriptionally regulated genes In standard experiments, profiling total mrna from cell pairs identifies genes being either transcriptionally induced (green) or repressed (red) during the transition. However, mrnas potentially regulated by translational control (grey circles / blue arrows) are not detected. TIBS 26,

14 Polysome-bound mrna expression profiling detects both, transcriptionally and translationally regulated genes II sucrose gradient fractionation, profiles of 4 compartments Fractionating total mrna into polysome-bound and ribosome-free mrna populations (blue arrows) enables detection of translational changes (bottom half). mrnas redistributing (blue squares) from ribosome-free towards polysome-bound pool (translational activation), or vice versa (translational repression), will display different hybridization signals. Note that hybridization signals for transcriptionally regulated mrnas (red, green) will be uncovered as in standard profiling.

15 Validation of Method I Translational control during differentiation of erythroid cells Joosten, M., Blazquez-Domingo, M., Lindeboom, F., Boulme, F., Van Hoven-Beijen, A., Habermann, B., Lowenberg, B., Beug, H., Müllner, E. W., Delwel, R., and Von Lindern, M.; Translational Control of Putative Protooncogene Nm23-M2 by Cytokines via Phosphoinositide 3-Kinase Signaling; J Biol Chem 279,

16 Validation of Method II Translational regulation during activation of T lymphocytes (A) Staining of the filter indicating the distribution of 28S, 18S, and 5S RNA. (B E) Sequential hybridisation of the filters with [alpha- 32 P]dCTP-labeled probes specific for ATF-4, transducinbeta2, Gadd 153, and p38 MAP kinase cdnas, respectively. F) Signals were quantified by laser densitometry. To facilitate comparison, total RNA content summarised over all fractions was set to 100%. Empty circles, distribution of mrna in resting T cells (R); filled circles, distribution of mrna in activated T cells (A). Mikulits, W., Pradet-Balade, B., Habermann, B., Beug, H., Garcia-Sanz, J.A., and Müllner, E.W. Isolation of translationally controlled mrnas by differential screening FASEB J. 14,

17 Protein abundance, not mrna abundance is conserved Considering orthologs across highly divergent species, abundances of proteins are more conserved than abundances of the corresponding mrnas, suggesting that protein abundances may be evolutionarily favored. (Numbers indicate Spearman rank correlation coefficients [?] between molecular abundances.) Data such as these support an important role for regulatory mechanisms occurring downstream from the setting of mrna levels. Vogel & Marcotte, Nat Rev Genet 13(4),

18 Key concepts for analysis of protein abundance I The Western blot-problem Vogel & Marcotte, Nat Rev Genet 13(4), Western blot-problem : How to compare abundance of two different proteins? Calibration curve for each one with pure (isolated or synthesized) protein

19 Key concepts for analysis of protein abundance II!! Western flow cytometry Vogel & Marcotte, Nat Rev Genet 13(4),

20 from this and much other work more than 50 % of all mrnas are subject to (individual! ) translational regulation or differential mrna stability or localization or Remainder of lecture: How?

21 Basics I Molecules Reminder; ~ 1st year BSc plus extension

22 Eukaryotic messenger RNA (mrna) Insult-Variant Das primäre Transkript erhält durch Übertragung von GTP und Methylierung von Guanin in Position 7 eine Cap-Struktur am 5'-Ende und einen poly-a-schwanz am 3'-Ende. Die so modifizierte RNA nennt man hnrna (heterogenous nuclear RNA) oder "pre-messenger- RNA". Diese reift im danach im Kern durch Splicing zur eigentlichen mrna, bei welcher die nicht-kodierenden Abschnitte (Introns) entfernt sind. Die mrna passiert die Kernporen und gelangt so ins Cytoplasma. Dort erfüllt sie ihren eigentlichen Zweck, sie wird übersetzt ("translatiert"). Die mrna ist Übermittler (Bote) der genetischen Information zwischen Kern und Cytoplasma: sie dient als Matrize für die Übersetzung von Nukleotidsequenzen in Aminosäurensequenzen. Translatiert wird aber nur derjenige Abschnitt der mrna, welcher vom Startcodon AUG bis zu einem Stopcodon (UAA, UAG oder UGA) reicht.

23 Initiation bei Prokaryoten Insult-Variant Prokaryoten können mehrere Proteine auf einer einzigen mrna codieren und somit gleichzeitig verschiedene Proteine von derselben mrna synthetisieren. Solche polycistronische mrnas benötigen deshalb auch dementsprechend viele Shine-Dalgarno-Sequenzen (S/D; 5'-AGGAGGU-3 ) und ebensoviele Start- und Stop-Codons: Der Translationsvorgang selbst beginnt, sobald eine Initiator-tRNA mit dem Start-Codon, das durch die 30S Untereinheit ausfindig gemacht wurde, eine Basenpaarung eingeht. Diese Initiator-tRNA ist bei Bakterien mit N- Formylmethionin (fmet) beladen. Den Komplex aus mrna, 30S Untereinheit und Formylmethionin-tRNA nennt man Initiationskomplex, und seine Bildung markiert das Ende der Initiationsphase der Translation. this will remain the only slide on prokaryotes

24 Initiation bei Eukaryoten Insult-Variant Der wesentliche Unterschied zu Prokaryoten betrifft die Art, wie die kleine Untereinheit des Ribosoms (40S bei Eukaryoten) an die mrna bindet und das AUG-Codon, an dem die Translation beginnen soll, ausfindig macht. Außerdem sind für die Initiation mindestens elf Initiationsfaktoren erforderlich, im Gegensatz zu E. coli, wo drei solche Proteine ausreichen. Bei Eukaryoten bindet einer dieser Initiationsfaktoren, das cap-bindende Protein eif4e, an die 5'- Cap-Struktur der mrna und fördert die Bildung eines Komplexes zwischen der mrna und der 40S Untereinheit des Ribosoms. Die 40S Untereinheit hat bereits Initiator-Methionyl-tRNA gebunden. Die 40S Untereinheit wandert dann der mrna entlang, bis sie ein Start-Codon erreicht. Dort wird die 60S Untereinheit des Ribosoms angefügt, und es resultiert ein 80S-Initiationskomplexes, der die Initiator-Met-tRNA Met sowie mrna enthält und für die Elongation bereit ist.

25 System components characterization of the transcriptome RNA sub-classes in a typical mammalian cell

26 Sidestep intron versus exon length Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig 6.32 functions for intron RNAs? long non-coding (lnc)rnas

27 System components ribosomal RNAs Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig

28 System components ribosomal RNAs Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig

29 System components ribosomes Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig 6.63

30 System components ribosomes en.wikipedia.org/wiki/ribosome contains animated GIFs of the structures Atomic structure of the 30S Subunit from Thermus thermophilus. Proteins in blue, the single RNA chain in orange. Atomic structure of the 50S Subunit from Haloarcula marismortui (Archebacterium). Proteins in blue, the two RNA chains in orange and yellow. The small patch of green in the center of the subunit is the active site.

31 System components trnas Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig 6.52

32 System components trnas + aminoacyl-trnas activation of amino acids by individual specific aminoacyl-trna-synthetases in consequence, RNA double strands contain G=U as well as A=U base pairs! Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig

33 System components mrnas G. Pesole et al., Gene 276, 73 81

34 System components polysomes The length of a eukaryotic mrna (2000 b on average), permits translation by several ribosomes simultaneously. Such ribosome-mrna (better: mrnp) complexes are called either poly-ribosomes or polysomes. This allows for rapid and efficient protein synthesis. Polysomes can be visualized by electron microscopy. To put the scale bar below into perspective: As can be seen, 4 ribosomes next to each other 0.1µm = 100 nm 1 ribosome = 25 nm. Average diameter of a cell is ~ 15 µm = nm a chain of 600 ribosomes reaches from end of a cell to the other. what about the size of mitochondria? Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig 6.76

35 System components bringing it all together into a RING Alberts, Molecular Biology of the Cell 5/e 2008 Wiley-VCH, Fig 6.76 Complexes formed on capped, polyadenylated double-stranded RNA in the presence of eif4g, poly(a)-binding protein (PABP) and eif4e but in the absence of ribosomes as visualized by atomic force microscopy. Nature Rev Mol Cell Biol 2,