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Kozak Consensus Sequence the Amount of Protein Synthesized from a Given Mrna Also Depends on the Strength of the So Called Kozak Sequence Around the AUG Start Codon

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Kozak Consensus Sequence the Amount of Protein Synthesized from a Given Mrna Also Depends on the Strength of the So Called Kozak Sequence Around the AUG Start Codon IRESs Internal ribosome entry sites Internal translation initiation via IRESs – textbook version Cit IRES-dependent translation, mainly observed in viruses, only need a subgroup of eIFs which are assembled at folded IRES-structures this view is too simplistic Internal ribosome entry sites – IRESs • discovery: RNAs of picornaviruses (e.g. poliovirus, encephalomyo-carditis virus (EMCV), foot and mouth disease virus (FMDV), hepatitis C virus (HCV) and rhinovirus) are not capped and have unusually long 5' untranslated regions (341 to more than 800 nucleotides). • their leader regions contain multiple AUG codons and their translation was not inhibited under conditions where cap structure-dependent translation is strongly reduced. • As of 2014, there were 60 animal and 8 plant viruses reported to contain IRES segments. Also >120 eukaryotic mRNAs are known to contain IRESs as well. • experiment: dicistronic mRNAs carrying the thymidine kinase (TK) and chlor- amphenicol acetyl transferase (CAT) open reading frames with the entire polio- virus leader sequence (P2) or parts of it were inserted between the two open reading frames. Measurement of second open reading frame translation in vivo and in vitro in the presence or absence of P2 and under conditions where the first open reading frame is not translated First historical experiments to detect internal initiation (A) Pelletier and Sonen- berg, Nature 334, 320- 325, (1988) (B) What is the virus’ rationale for the use of IRESs? TK = thymidine kinase, CAT = chloramphenicol- acetyltransferase used as selectable mar- kers before GFP et al. (A) Schematic representation of the bi-cistronic constructs used by Pelletier and Sonenberg. (B) In vivo translation in uninfected and poliovirus-infected COS-1 cells. The results of these experiments demonstrated the existence of internal initiation and its dependence on about 450 nucleotides of P2 sequence Experimental verification of IRES function Standard bi-cistronic test for in vitro assays to detect IRES elements The first ORF starts via standard Cap-dependent initiation and ends in a triple stop codon to ensure inhibition of eventual read-through (which CAN happen!) One fre- quently used reporter encoded by this part of the transcript is firefly luciferase. The second ORF is followed by the putative IRED domain, which itself is then followed by a 2nd ORF. The latter one frequently encodes renilla luciferase. The first ‘standard’ luciferase expression serves as control to normalize between experiments Example: Polio virus secondary structure of leader sequence and genome organization Genomic structure of poliovirus type 1 (Mahoney). The PV genome consists of a single-stranded, positive-sense polarity RNA molecule, which encodes a single poly- protein. The 5' non-translated region (NTR) harbors two functional domains, the cloverleaf and the internal ribosome entry site (IRES), and is covalently linked to the viral protein VPg at the 5’ end. De Jesus NH (2007). "Epidemics to eradication: the modern history of poliomyelitis". Virol. J. 4: 70. Type II IRESs (‘viral’) directly bind 40S ribosomal subunits in a way that their initiator codons are located in the ribosomal P-site without mRNA scanning. In general, internal initiation via a type II IRES has a similar translation factor requirement (among others, eIF-4G !) like cap-dependent translation initiation. They do not, however, require eIF1, 1A, 4B, and 4E. Full functionality in vitro in a fully reconstituted system was first shown for encephalo-myocarditis virus (EMC virus) IRES. Type II IRESs are found among others in Hepatitis C Virus-related viruses (of course in HCV itself), Poliovirus, Picornaviridae, Encephalo Myocarditis Virus (EMCV), Foot and Mouth Disease Virus (FMDV). In one subgroup of type II IRESs, namely those in picornaviridae (= rhinoviruses, enteroviruses) 43S ribosomal subunits do not bind directly to the IRESs but rather through high-affinity eIF4G-binding sites. This interaction is enhanced by eIF-4A. Hellen CU, Sarnow P (2001). "Internal ribosome entry sites in eukaryotic mRNA molecules". Genes Dev. 15, 1593–1612. Jackson, R.J., Hellen, C.U., and Pestova, T.V. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11, 113-127. Type II IRES structures Poliovirus, IRES ~ 450 b EMCV, IRES ~ 450 b Hence, IRES-mediated initiation is resistant to cellular regulatory HCV mechanisms, such as eIF2 IRES ~ 300 b phosphorylation and/or eIF4E sequestration Jackson, R.J., Hellen, C.U., and Pestova, T.V. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11, 113-127. IRES-structures are highly variable Structure prediction programs fail to detect functional IRES domains, this still has to be done experimentally. hepaptitis A virus IRES hepatitis C virus IRES figures: Wikipedia Most type II IRESs associate with additional RNA-binding proteins Foot and Mouth Disease Virus PTB poly-pyrimidin-tract binding protein PCBP2 poly-cytidine-tract binding protein 2 Both proteins are standard components of messenger- ribonucleoprotein complexes but normally not involved in translation initiation! Curr. Op. Biotech. 10, 458-464, (1999) Type I IRESs – the eukaryotic cell variant Some viral IRESs (herpesviridae, cripaviridae, Marek’s disease virus) as well as all cellular IRESs also require additional proteins to mediate their function. These are collectively known as IRES trans-acting factors (ITAFs). The role of ITAFs in IRES function is still subject to intense research (2017). Models for ribosome recruitment by IRES - eIFITAF complexes (a) The ITAF could stabilize the active conformation of the IRES RNA, which is bound by eIFs that interact with the ribosome, but without direct interaction between the IRES RNA and the ribosome. (b) The ITAF and eIFs could both interact directly with the ribosome, again with no direct IRES RNA–ribosome interactions. (c) The ITAF could stabilize the active conformation of the IRES RNAs, and both the IRES and eIFs could contact the ribosome. (d) The ITAF, the eIFs and the RNA could all directly contact the ribosome. Note that other combina- tions could occur and that these are not mutually exclusive possibilities. nobody knows Filbin & Kieft, Toward a structural understanding of IRES RNA function Curr Op Struct Biol (19), pp 267–276. TYPE I IRES-structures also are highly variable Structure prediction programs fail to detect functional IRES domains, this still has to be done experimentally. IRES in fibroblast growth factor I (FGF I) IRES in FGF II figures: Wikipedia IRES-dependent translation initiation of cellular mRNAs The list of cellular mRNAs that are thought to contain IRESs is growing (at present ~ 120), although a recent stringent test has questioned some of these claims. Cellular IRESs show little structural relationship to each other and their underlying mechanism remains largely unknown but probably follows the picornavirus paradigm of binding the eIF4G–eIF4A complex. Importantly, cellular IRES-containing mRNAs can also be translated by the scanning mechanism (c-Myc!), which raises the crucial question of what regulates the switch between these modes of initiation. One key parameter might be the intracellular concentration of eIF4G. The concentration of eIF4G (but not eIF4E !) is highly elevated e.g. in many advanced breast cancers, and in inflammatory breast cancer this results in efficient IRES- dependent translation of p120 catenin and vascular endothelial growth factor (VEGF) mRNAs. eIF-4E overexpression + cancer : Dolznig, TransCon II In other cancer cell lines with high eIF4G levels, overexpression of eIF4E-binding protein 1 (4E-BP1), to sequester eIF4E, coupled with hypoxia, activates VEGF and hypoxia-inducible factor 1α (HIF1A) IRESs. Another parameter that may determine which mechanism predominates is the intracellular concentration of ITAFs. Jackson et al. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11, 113-127. IRES-dependent translation initiation of cellular mRNAs Protein type Proteins Fibroblast growth factor (see figure on FGF-1 IRES versus FGF-2 IRES), Platelet-derived growth Growth factors factor B (PDGF/c-sis IRES), Vascular endothelial growth factor (VEGF IRES),Insulin-like growth factor 2 (IGF-II IRES) Antennapedia, Ultrabithorax , MYT-2 , NF-κB repressing factor NRF, AML1/RUNX1 , Gtx Transcription factors homeodomain protein Eukaryotic initiation factor 4G (elF4G !!), Eukaryotic initiation factor 4Gl (elF4Gl),Death associated Translation factors protein 5 (DAP5) Oncogenes ?! c-myc, L-myc, Pim-1, Protein kinase p58PITSLRE, p53 oncogenes vs. proto-ocncogenes Transporters/receptors Cationic amino acid transporter Cat-1, Nuclear form of Notch 2, Voltage-gated potassium channel Activators of apoptosis Apoptotic protease activating factor (Apaf-1) Inhibitors of apoptosis X-linked inhibitor of apoptosis (XIAP), HIAP2, Bcl-xL, Bcl-2 Proteins localized in Activity-regulated cytoskeletal protein (ARC), α-subunit of calcium calmodulin dependent kinase II neuronal dendrites dendrin, Microtubule-associated protein 2 (MAP2),neurogranin (RC3), Amyloid precursor protein Immunoglobulin heavy chain binding protein (BiP), Heat shock protein 70 (!), β-subunit of Others mitochondrial H+-ATP synthase, Ornithine decarboxylase (ODC), connexins 32 and43,hypoxia- inducible factor 1α (HIF-1α), adenomatous polyposis coli (APC) table modified from Wikipedia Mechanisms
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