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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 chloramphenicol acetyl transferase (CAT) open reading frames with the entire poliovirus 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

(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

First historical experiments to detect internal initiation (A)

(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.

Pelletier and Sonen-berg, Nature 334, 320-325, (1988)

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

Experimental verification of IRES function

Standard bi-cistronic test for in vitro assays to detect IRES elements

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.

Example: Polio virus secondary structure of leader sequence and genome organization

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

Hence, IRES-mediated initiation is resistant to cellular regulatory mechanisms, such as eIF2 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.

Poliovirus, IRES ~ 450 b

HCV IRES ~ 300 b

EMCV, IRES ~ 450 b

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

Curr. Op. Biotech. 10, 458-464, (1999)

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!

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

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

Growth factors Fibroblast growth factor (see figure on FGF-1 IRES versus FGF-2 IRES), Platelet-derived growth factor B (PDGF/c-sis IRES), Vascular endothelial growth factor (VEGF IRES),Insulin-like growth factor 2 (IGF-II IRES)

Transcription factors Antennapedia, Ultrabithorax , MYT-2 , NF-κB repressing factor NRF, AML1/RUNX1 , Gtx homeodomain protein

Translation factors Eukaryotic initiation factor 4G (elF4G !!), Eukaryotic initiation factor 4Gl (elF4Gl),Death associated 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 neuronal dendrites

Activity-regulated cytoskeletal protein (ARC), α-subunit of calcium calmodulin dependent kinase II dendrin, Microtubule-associated protein 2 (MAP2),neurogranin (RC3), Amyloid precursor protein

Others Immunoglobulin heavy chain binding protein (BiP), Heat shock protein 70 (!), β-subunit of mitochondrial H+-ATP synthase, Ornithine decarboxylase (ODC), connexins 32 and43,hypoxia-inducible factor 1α (HIF-1α), adenomatous polyposis coli (APC)

IRES-dependent translation initiation of cellular mRNAs

table modified from Wikipedia

Mechanisms of cap-dependent versus IRES-dependent translation initiation

Factor X = ITAFs Sonenberg, Trends Genet (16), pp 469-473

first reference of connection

to apoptosis

new factor(s)

Mechanisms of cap-dependent (left) and IRES-dependent (right) translation initiation

Figure legend

Conditions and initiation factors thought to favour one mode of translation initiation over the other are shown in three separate panels. Only eukaryotic initiation factors (eIF) pertinent to this article are indicated. The cap-binding protein eIF-4E (light blue) recognises and binds to the m 7´me-GpppX cap structure (red). The capped end of the mRNA is bridged with the 40S ribosomal subunit (green) by an adapter molecule eIF-4G (purple). The binding of eIF-4G to the 40S subunit is facilitated by eIF-3 (yellow). EIF-4A (dark blue) is an RNA-dependent ATPase and RNA helicase involved in the unwinding of the secondary structure of the untranslated region (UTR). Factor X represents a putative protein (or proteins) binding to internal ribosome entry site (IRES) elements that has been suggested by several laboratories to be involved in internal initiation. Asterisks denote initiation factors that have been proteolytically modified (indicated by scissors) by viral protease (2A-pro), caspase 3 (Casp3) or other caspases (Casp).

Trends Genet (16), pp469-473

a | The cap-binding protein, eukaryotic initiation factor-4E (eIF4E; blue), binds to the 5’m7GpppN cap structure (red). The capped end is bridged to the 40S ribosomal subunit (green) by the adapter molecule eIF4G (dark green), which also binds to eIF3 (yellow). eIF4A (cyan) is an RNA-dependent ATPase and RNA helicase thought to be involved in the unwinding of the secondary structure of the mRNA 5’untranslated region (UTR). Poly(A)-binding protein (PABP; pink) circularizes the mRNA through interaction with both 3’UTR (through the poly(A) tail) and 5’UTR (through interaction with eIF4G).

b | Internal ribosome-entry site (IRES) trans-acting factors (ITAFs; orange) and proteolytic fragments of eIF4GI or p97/DAP5/NAT1, a distant homologue of eIF4G, (light grey) stimulate IRES translation. Only eIFs pertinent to this process are indicated.

Hokik and Sonenberg, Nat. Rev. Mol. Cell. Biol. 6, 318-327, (2005)

Cap-dependent versus IRES-dependent translation initiation of cellular mRNA similar to previous but evolved

eiF4G is the prototype within a small gene family: binding partners of interactive domains are shown above eIF4-GI. Domains are indicated by the same colour for all three proteins. The similarity of the eIF-3/eIF-4A binding regions to those of eIF4-GI is indicated. Poliovirus 2A protease and caspase (Casp) cleavage sites are indicated by arrows.

Trends Genet 16, 469-473, (2000)

Mammalian eIF4G clevage in viral infection and apoptosis

Representation of the longest isoform of eIF4G, its p100 (carboxy-terminal two-thirds), its p50 (central one-third) fragments, and of p97, showing binding sites for SLBP-interacting protein 1 (SLIP1), poly(A)-binding protein (PABP), eIF4E, eIF4A, eIF3 and MAP kinase interacting Ser/Thr kinase 1 (MNK1) or MNK2 and for RNA (dotted lines below eIF4GI). Interactions of eIF4GI with eIF4E and MNK1 are required for phosphorylation of eIF4E by MNK1. Interactions of eIF4GI with PABP and SLIP1 tether eIF4GI to the mRNA’s 3 ′ end. The amino acid residues at the amino-termini of the PABP-binding domain (PAM1), eIF4E-binding domain (4E-BR) and HEAT1 (also known as MIF4G), HEAT2+3 domains are indicated as is the cleavage site in eIF4GI for picornavirus proteinase 2Apro; N-terminal domain binds eIF4E and PABP, C-terminal domain provides functions of eIF4GI required for initiation on type 1 and type 2 IRESs

~ same as previous, more detail with respect to functional proteolytic eIF4G1 fragments Jackson et al. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol (11), pp113-127.

Mammalian eIF4G cleavage

Internal ribosome initiation of translation and the control of cell death The majority of cellular stress conditions lead to inhibition of cap-dependent transla-tion. Some mRNAs, however, are translated by a cap-independent mechanism, me-diated by ribosome binding to IRES elements located in the untranslated region. Inter-estingly, IRES elements are found in the mRNAs of several survival factors, onco-genes as well as proteins crucially involved in the control of apoptosis. These mRNAs are translated under a variety of stress conditions, including hypoxia, serum depri-vation, irradiation and apoptosis. Thus, IRES-mediated translational control might have evolved to regulate cellular responses in acute but transient stress conditions that would otherwise lead to cell death.

Martin Holcik, Nahum Sonenberg, and Robert G. Korneluk Trends Genet (16), pp469-473 ------------------------------------------------------- A second main function of cellular IRES mRNAs is to produce proteins required for mitosis, during which phosphorylation of eIF-4E is massively reduced, resulting in low efficiency of cap-dependent translation.

IRES-dependent translational regulation and cell death

Stages leading to cell death in red, pro-survival events in green.

Trends Genet (16), pp469-473

As of September 2009, there were 115 non-viral mRNAs, 60 animal and 8 plant viruses reported to contain ‘proven’ IRESs

Mokrejs et al.; Nucleic Acids Res. 34 (Database issue): D125–30.

Hokik and Sonenberg, Nat Rev Mol Cell Biol 6(4), pp318-327

IRES-dependent apoptotic pathways and their regulation

survival

death

Figure legend (modified): Apoptotic signals converge on caspases in a self-amplifying cascade. Apoptotic triggers can be external or internal, and evoke distinct cellular responses.

Intracellular stress (such as DNA damage) results in activation of the mitochondrial (or intrinsic) pathway. This is characterized by Cytochrome c release, formation of the apoptosome (cytochrome c, pro-apoptotic protease-activating factor-1 (APAF1) and pro-caspase-9) and caspase-9 activation.

Endoplasmic reticulum (ER) stress results in the activation of an ER-specific pathway as well as the mitochondria-dependent pathway. The ER-specific pathway activates caspase-12 in mice (or a similar caspase in humans) and/or caspase-4, and subsequently, caspase-3 or caspase-7.

Extracellular ligand binding to death receptors like FAS-L triggers the extrinsic pathways that either directly result in caspase activation, or require further amplification through the mitochondrial pathway (dashed arrow), depending on the cell type

All apoptotic signals converge at effector caspases, such as caspase-3 and caspase-7. Control points along these pathways regulate release of cytochrome c and other apoptogenic factors from mitochondria by BCL2 family proteins like BAX and BAK. Others regulate the levels or activity of caspase inhibitors, or inhibitors of apoptosis (IAPs), such as X-chromosome-linked inhibitor of apoptosis (XIAP), through their antagonists (such as SMAC/DIABLO or serine protease HTRA2/OMI).

BCL2, β-cell leukaemia/lymphoma-2; FADD, Fas-associated death domain.


IRES-dependent apoptotic pathways and their regulation

IRES-dependent regulation of XIAP mRNA in apoptosis

Triggering of apoptosis results in activation of caspases that cleave numerous cellular substrates. Among caspase targets are initiation factors, including members of the eukaryotic initiation factor-4G (eIF4G) family. Their cleavage leads to inhibition of cap-dependent protein synthesis that often accompanies apoptosis.

a | By contrast, the translation of caspase inhibitor, X-chromosome-linked inhibitor of apoptosis (XIAP), is mediated by an internal ribosome-entry site (IRES) that is induced by certain apoptotic conditions, such as serum starvation or low-dose γ-irradiation, which results in the suppression of caspases to delay the commitment of cells to apoptosis. Once the stress trigger is removed, the cells may restore their metabolic activity and resume growth.

Hokik and Sonenberg, Nat Rev Mol Cell Biol 6(4), 318-327, (2005)

b | Other types of stress, such as Fas- or etoposide-induced apoptosis, favor the IRES-mediated translation of pro-death proteins, such as the pro-apoptotic protease-activating factor-1 (APAF1). The caspase-mediated processing of eIF4GI and the distant homologue of the eIF4G eukaryotic initiation factor p97/DAP5/NAT1 produces specific cleavage products (middle terminal fragment of apoptotic cleavage of eIF4G (M-FAG) and p86/ DAP5) that selectively enhance the translation of the APAF1 IRES, which results in increased levels of APAF1 protein to maintain and further accelerate cell death.

C-FAG, carboxy-terminal fragment of apoptotic cleavage of eIF4G; m7G, cap structure; N-FAG, amino-terminal fragment of apoptotic cleavage of eIF4G.


IRES-dependent regulation of APAF mRNA in apoptosis

TOP mRNAs Top = terminal oligopyrimidine tract

TOP mRNAs

TOP mRNAs carry a terminal oligo-pyrimidine tract (TOP) adjacent to the cap structure in their 5' untranslated region. The oligo-pyrimidine tract apparently serves as a protein binding site and protein binding inhibits translation.

TOP mRNAs encode proteins of the translational apparatus (including ribosomal proteins, elongation factors and poly(A)-binding protein) which are not translated in resting cells and shift into polysomes when cells are stimulated to grow.

Correlation between enhanced translation of TOP mRNAs and S6 phosphorylation was noted. These data are still debated since the molecular mechanism by which the phosphorylated protein might enhance translation remains obscure (2013).

(a) Comparison of the consensus sequence of the first 16 nucleotides in TOP mRNAs from frog, mouse and human (9, 88 and 89 mRNAs, respectively). The proportion of each nucleotide is depicted by the relative height of the letters A, C, G and U.

Translational repression of TOP mRNAs – sequence 'homology'

Ruvinsky, I., and Meyuhas, O.; Trends Biochem Sci 31, 342-348, (2006)

One specific example: TOP-motif of mouse ribosomal mRNA S16 (= rpS16; nomenclature = mRNA for the 16th-largest protein of the small ribosomal subunit). Wikipedia

TOP mRNAs and translational control I Synthesis of many proteins associated with the translational apparatus of higher euka-ryotes, including all ribosomal proteins, elongation factors 1A, 1B and 2, and poly(A)-binding protein, is selectively regulated at the translational level. The corresponding mRNAs are characterized by the presence of a 5’-terminal oligopyrimidine tract (5’ TOP) and are therefore referred to as ‘TOP mRNAs’ (see Figure Ia, next slide). This structural motif comprises the core of the translational cis-regulatory element of these mRNAs and invariably consists of a cytosine at the cap site, followed by an uninterrupted stretch of 4-15 pyrimidine bases.

The composition of the 5’ TOP, although varying among TOP mRNAs even within the same species, maintains, in most cases, a similar proportion of cytosine and uracil nu-cleotides. The translational control of TOP mRNAs is manifested by a selective unloa-ding of these mRNAs from polysomes (translational repression) when progression through the cell cycle is blocked at the G0, G1/S or G2/M phase and when cells are deprived of amino acids (Figure Ib, see 2nd-next slide).

Conversely, TOP mRNAs are recruited into polysomes (translational activation) when resting cells are induced to proliferate or to grow (increase in size), or when cells starved of amino acids are re-fed.

Ribosomal protein S6 phosphorylation: from protein synthesis to cell size Ruvinsky, I., and Meyuhas, O.; Trends Biochem Sci 31, pp342-348

(b) TOP mRNAs are unloaded from polysomes (translationally repressed) on (i) amino acid starvation or (ii) when cell-cycle progression is blocked at G1 by serum starvation, (iii) contact inhibition or (iv) terminal differentiation; (v) at G1/S by inhibition of DNA synthesis, with hydroxyurea or aphidicolin; or (vi) at G2/M by inhibition of mitotic spindle assembly by nocodazole, an inhibitor of microtubule polymerization.

Translational repression of TOP mRNAs III

Ruvinsky and Meyuhas, Trends Biochem Sci 31, pp342-348

Cytoplasmic extracts from rat pheochromocytoma (PC12), mouse NIH 3T3, and human HeLa cells either untreated (control) or arrested by serum starvation, treated by hydroxyurea, or nocodazole respectively. Extracts were centrifuged through sucrose gradients and separated into polysomal (P) and subpolysomal (S) fractions. RNA was analyzed by a TOP (rpL32) or non-TOP (actin) probe.

Meyuhas & Dreazen, Ribosomal protein S6 kinase: From TOP mRNAs to cell size. Progr Mol Biol Translat Sci 90 (Translational Control in Health and Disease), Chapter 3, pp109-153, (2009)

Translational repression of TOP mRNAs IV Arrest of the cell-cycle progression results in translational repression of TOP mRNAs

A principal effector downstream of mTOR is the S6K1 serine/threonine kinase. After receiving a proliferative signal mediated by PI3K/Akt, mTOR phosphorylates and activates S6K1. In turn, S6K1 phosphorylates and activates the 40S ribosomal S6 protein, facilitating recruitment of the 40S ribosomal subunit into actively translating polysomes. In particular, translation of mRNAs with a 5´TOP is enhanced. These 5´TOP mRNAs code primarily for ribosomal proteins, elongation factors, and insulin-like growth factor-II. Dephosphorylation of S6K1 decreases synthesis of components of the protein translation system and results in a profound decrease in protein synthesis.

In reality, regulation of S6K1 activity is more complicated

http://www.medscape.org/viewarticle/494951_2 --> Jefferies et al. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. Embo J. 1997;16:3693-3704. Terada et al. Rapamycin inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci U S A. 1994;91:11477-11481.

mTOR activation of S6K Translation of TOP mRNAs V

mTORC1

Is -this- the real reason for the complicated S6K system?

M Kozak Codon bias

Kozak consensus sequence

Sequence logo derived from translation initiation regions of all human genes. There are significant differences between groups of eukaryotes. Each letter is written in proportion to its biased use. If a base were used in all ~25,000 genes it would score 2 and be written two units tall. The most significantly biased bases are -3 (A) and +4 (G). AUG not drawn to scale (and would score 2 bits of information on this scale; a sequence logo (Wiki) is derived differently than the diagram on TOP mRNAs and involves thermodynamic parameters).

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. Marilyn Kozak, Nature 308, 241–246, (1984)

or (gcc)gccRccATGG

http://upload.wikimedia.org/wikipedia/commons/b/bf/KozakConsensus.jpg�

strong consensus: the nucleotides at positions +4 (i.e. G in the consensus) and -3 (i.e. either A or G in the consensus) relative to the number 1 nucleotide must both match the consensus (there is no number 0 position).

'adequate' consensus: has only 1 of these sites

'weak' consensus: has neither. The cc at -1 and -2 are not as conserved, but contribute to the overall strength. There is also evidence that a G in the -6 position is important in the initiation of translation.

The first mutation found in the Kozak sequence was GC at -6 of the human β-globin gene which disrupted its function. It was found in a family from Italy suffering from tha-lassaemia intermedia.

Br J Haematol 124, 224–231, (2004)

Kozak consensus sequence

Codon bias – within and between genomes

Which of the 60 possible codons are actually used? Relevance for bio-technology et al. Plotkin & Kudla, NatRevGenet 12, 32-42, (2011)

Codon bias within and between genomes

Relative synonymous codon usage (RSCU) plotted for 50 randomly selected genes from each of nine species. RSCU ranges from 0 (when the codon is absent), through 1 (when there is no bias) to 6 (when a single codon is used in a six-codon family). Methionine, tryptophan and stop codons are omitted. Note the extensive heterogeneity of codon usage among human genes.

Plotkin & Kudla, NatRevGenet 12, 32-42, (2011)

uORFs upstream open reading frames

uORFS modulate translation efficiency Significance –uORFs are important control elements of growth and differentiation

* almost 10 percent of vertebrate mRNA contain one or more uORFs

* about 2/3 of mRNAs encoding vertebrate growth regulatory proteins have uORFs

Morris and Geballe, Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol 20, 8635-8642.

1) initiating translation at proximal AUG 2) or a distal downstream AUG 3) stalling 4) altering mRNA stability 5) dissociation from mRNA

What can happen when machinery encounters an uORF? (A) Leaky scanning is dependent on the efficiency of uAUG recognition; sometimes the ribosome can translate the uORF, but other times the scanning machinery bypasses the uAUG, recognizing the downstream AUG and translating the main ORF (B) When a scanning ribosome recognizes and translates a functional uORF, a small peptide is translated; if termination of the uORF is efficient, 60S and 40S ribosomal subunits dissociate from the transcript and the main ORF is not translated. (C) A uORF can repress translation of the main ORF in a peptide-dependent manner; in this case, the uORF-encoded peptide interacts with the translation machinery and promotes ribosome blockage.

Barbosa C, Peixeiro I, Romão L (2013). Gene Expres-sion Regulation by Upstream Open Reading Frames and Human Disease. PLoS Genet 9, e1003529. doi:10.1371/journal.pgen.1003529

(D) The termination codon of a uORF can be recognized as premature and nonsense-mediated mRNA decay (NMD) is triggered through a mechanism involving the UPF1 protein and ribonucleases (more in lecture TransCon II, SuSe

(E) After translation termination of the uORF, the 40S ribosomal subunit can remain associated with the transcript, resume scanning, and recognize the downstream main AUG—a mechanism designated as translation reinitiation.

Barbosa et al, PLoS Genet 9(8): e1003529.

RNA Stability TransCon II

rare

What can happen when machinery encounters an uORF?

(G) In response to stress, the presence of more than one uORF in a transcript can promote an increase in translation efficiency of the main ORF; reinitiation after translation of the uORF1 is less efficient since there is less ternary complex available. Consequently, reinitiation will take more time/distance to occur and the ternary complex will only be available by the time the 40S ribosomal subunit has already bypassed the subsequent uORFs, augmenting the recognition of the main AUG. GCN4 + ATF4 a little later (H) In response to stress, the presence of one uORF in a transcript can promote an increase of the corresponding protein levels; the higher levels of phosphorylated eIF2α contribute to increase leaky scanning of the uORF and translation of the main ORF is favored.


rare

What can happen when machinery encounters an uORF?

factor affecting effects of uORFs

(F) The impact that the uORFs can have on translation depends on (i) distance between the 5′ cap (m7G) and the uORF (distance to the cap) (ii) context in which the uORF AUG is located (AUG context / Kozak consensus, later) (iii) length of the uORF (iv) number of uORFs per transcript (v) secondary structure of the uORF (vi) conservation among species (vii) length of the intercistronic sequence(s) (viii) position of the uORF termination codon, upstream or downstream of the main initiation codon (length, number, secondary structure, conservation, position of stop codon).

The increase of translational repression exerted by a uORF correlates with increasing distance between the m7G and the uORF, increasing length of the uORF and intercistronic sequence, a higher number of uORFs, and a stronger uAUG Kozak context.


Abundance of uORFs

Found in 10 percent of all vertebrate mRNAs examples of struc-tural organization length of 5’UTR in nucleotides (nts)

boxes with numbers: uORF(s), number indicates corresponding length in codons. Barbosa et al, PLoS Genet 9(8): e1003529.

Regulation of exemplary mRNAs with uORFs

1 AdoMetDC S-adenosylmethionine decarboxylase

2 Arg2 arginine-specific carbamoyl phosphate synthetase

3 GCN4 yeast transcription factor for amino acid biosynthesis later: ATF 4+5 mammalian activating transcription factor-4 uORFs depicted by thick horizontal lines

1)

3)

2)

Regulation of exemplary mRNAs with uORFs 1) Mammalian AdoMetDC (= S-adenosylmethionine decarboxylase)

catalyzes a key step in the biosynthesis of the polyamines spermidine and spermine

2) Neurospora arg-2: The small subunit of the arginine-specific carbamoyl

phosphate synthetase (CPA) 3) Translational control of the yeast GCN4 gene is the best understood

example of regulatory interactions of multiple uORFs. GCN4 encodes a transcription factor that activates the expression of approximately 50 genes of amino acid biosynthesis. During amino acid starvation, general protein synthesis is inhibited and translation of GCN4 mRNA is markedly enhanced.

4) ATF 4+5 (mammalian activating transcription factor-4); similar to the

regulation of GCN4; uORF2 overlaps with the ATF4 ORF, thus the translation of uORF2 suppresses the translation of ATF4.

Peptoswitches are cis-acting regulatory short peptides encoded by short open reading frames (sPEPs) that respond to specific small molecules in the cellular environment. These small molecules interact, either directly or through an intermediary, with the nascent sPEPs (indicated by the dashed arrow) to stall ribosomes at the upstream open reading frame (uORF), which prevents their progression and inhibits translation of the downstream coding DNA sequence (CDS). Shea and Rothnagel, Nat Rev Genet 15, 193-204, (2014)

1) AdoMetDC, arg-2 – Peptoswitch

1) Translational control of AdoMetDC (= S-adenosylmethionine decarboxylase AdoMetDC is an enzyme of the polyamine biosynthesis pathway (metabolic function). Its expression is repressed at the translational level by spermidine and spermine.

AdoMetDC mRNA carries a short uORF encoding the peptide MAGDIS, which interacts with spermidine and spermine. This leads to inhibition of peptidyl-tRNA hydrolysis, ribosome stalling and inhibition of AdoMetDC ORF translation. At low polyamine concentrations, 40S subunits bypassing the uORF AUG continue scanning until encountering the ‘real’ start codon.

G. Lynn et al., J. Biol. Chem. 276, pp 38036-38043

A Raney et al., J. Biol Chem 277, 5988ff

The polykation (polyamin) spermin is involved in metabolic activities and found in ribosomes as well as in sperm, in both latter cases stabilizing nucleic acid structure

Model: 80S ribosome with newly synthesized attenuator peptide (AAP) which interacts with arginine and stalls at the uORF termination codon. This blocks scanning of other 40S initiation complexes which bypassed the AUG of the uORF and thus would have initiated at the AUG of the main ORF. Main feature in cases 1) + 2): there is a FUNCTION of the short peptide from the ORF

2) Translational control of Neurospora arg-2 = small subunit of the arginine-specific carbamoyl phosphate synthetase, CPA

High levels of arginine lead to ribosome stalling. This results from the 24 amino acid attenuator peptide encoded by the uORF. It works even when the peptide is fused to a reporter protein. The amino acid sequence of the attenuator peptide is important.

sPEPs – short peptides encoded by short open reading frames

Shea and Rothnagel, Nat Rev Genet 15, 193-204, (2014)

Translation of the YEAST transcriptional activator GCN4 is regulated by 4 uORFs. At low le-vels of eukaryotic initiation factor-2α (eIF2α) phosphorylation, when ternary complex is abun-dant, ribosomes initiate at uORF1 or continue scanning to initiate at uORF2, or uORF3 or uORF4. This, step by step strongly decreases the probability of initiation at the ‘real’ AUG. By contrast, during amino-acid starvation, increased levels of eIF2α phosphorylation lower the abundance of the ternary complex and initiation at uORF1-4 becomes less frequent, which allows scanning ribosomes to reach the GCN4 ORF.

Coding regions, green rectangles; uORFs, pink rectangles; 5’UTRs, thin lines; initiation codons (AUG), arrows; ribosomes in orange (60S subunit, dark; 40S subunit, light); m7G, cap.

3) Translational control of

GCN4

via uORFs

THE ‘classical’ example in most textbooks Hokik and Sonenberg, Nat. Rev. Mol. Cell. Biol. 6(4), 318-327, (2005)

ternary complex = eIF2, Met-tRNA, GTP

5'TRU: Identification and analysis of translationally regulative 5‘ untranslated regions in amino acid starved yeast cells Rachfall, Heinemeyer et al., Mol Cell Proteomics. 2011 Mar 28 http://www.ncbi.nlm.nih.gov/pubmed/21444722?dopt=Abstract in addition to GCN4 there are additional translationally modulated

amino acid metabolism mRNAs Consequence in general: Also ‘genuine’ start codons will not always lead to proper initiation what will happen? Kozak consensus sequence

Ernst Müllner, 2011-11

Translation of the mammalian activating transcription factor-4 (ATF4) is regulated by two uORFs. Similar to GCN4, when the ternary complex is abundant (in the presence of low levels of eIF2α phosphorylation), ribosomes initiate at uORF1 or uORF2. As uORF2 overlaps with the ATF4 ORF, the translation of uORF2 fully suppresses translation of ATF4. During endoplasmic reticulum (ER) stress, when the levels of ternary complex are reduced, ribosomes may scan through uORF2 and initiate at the ATF4 initiation codon.

examples 3) + 4): the sequence within the short ORF is irrelevant

4) Translational control of

ATF4

via uORFs similar to GCN4, another example of the ‘major’ mechanism Hokik and Sonenberg, Nat. Rev. Mol. Cell. Biol. 6(4), pp318-327


Jackson et al. Nat Rev Mol Cell Biol 11, 113-127, 2010

4) Translational control of ATF4 via uORFs and stress

structure

normal

stress

Legend to previous

a | Size, spacing and position of the two upstream open reading frames (uORFs) in human, mouse, rat, cow and chicken activating transcription factor 4 (ATF4) mRNAs and the four mammalian ATF5 mRNAs.

b | Translation in unstressed conditions, when eIF2–GTP–MetRNA ternary complexes are abundant. Small (40S) ribosomal subunits, with associated eIF2-TCs (blue), scan the mRNA. Nascent protein chains are shown by the black zigzag line associated with the large (60S) ribosomal subunit. If eIF2-TCs are abundant, most of the 40S subunits that resume scanning after uORF1 translation will acquire a new eIF2-TC in time to initiate translation of uORF2, and ribosomes that translate this second uORF will be unable to initiate at the ATF4 or ATF5 AUG because uORF2 is too long to allow rescanning.

c | Translation in stressed conditions, when eIF2-TC availability is low owing to eIF2 phosphorylation e.g. by activated PKR-like endoplasmic reticulum kinase (PERK). Consequently, most of the 40S subunits that resume scanning after translating uORF1 acquire a new eIF2-TC only after they have migrated past the uORF2 initiation codon, but in time to initiate at the next AUG, which is at the start of the ATF ORF in both cases.

4) Translational control of ATF4 via uORFs

Jackson et al. Nat Rev Mol Cell Biol 11, 113-127, 2010

Before It Gets Started: Regulating Translation at the 5 ′ UTR

Araujo, Patricia R.; Yoon, Kihoon; Ko, Daijin; Smith, Andrew D.; Qiao, Mei; Suresh, Uthra; Burns, Suzanne C.; Penalva, Luiz O. F. Comparative and Functional Genomics Vol 2012 (2012) doi:10.1155/2012/475731

Cit: ‘Only a small fraction of the posttranscriptional regulatory elements located in human 5′ UTRs have been characterized yet’. Those identified UTR elements are catalogued in a web-resource maintained by Graziano Pesole’s group called UTRdb - http://utrdb.ba.itb.cnr.it/’

Control of eukaryotic protein synthesis by upstream open reading frames in the 5’-untranslated region of an mRNA; Meijer and Thomas, Biochem. J. 367, 1-11, (2002)

cf. Kozak consensus sequence

Mutations/polymorphisms in uORFs and disease just for reading






Barbosa et al, PLoS Genet 9(8): e1003529. Ernst Müllner, 2014-01

Preview TransCon II

• proteins binding selectively to specific mRNAs

• mRNA 3’ end formation + polyadenyltion alternative polyadenylation ( alternative splicing) re-adenylation in development

• mRNA localization in development in somatic cells

• mRNA stability – general and mRNA (class) specific stress granules and P bodies (short visit to shRNAs, miRs)

• (rare) diseases via aberrations in translational machinery ribosomopathies altered tRNAs

• translational control and cancer – dozens of evidences tumor type (Helmut Dolznig) mechanistically (miRs, initiation factors, processing, ...)

ribosome biogenesis, mRNA editing, lncRNAs, nuclear export, ...

Preview TransCon II

Before It Gets Started: Regulating Translation at the 5 ′ UTR Araujo et al, Comp Funct Genomics 2012 (2012) doi:10.1155/2012/475731 Cit: RBPs can have antagonistic functions when regulating translation. An interesting example is the regulation of p21 in the context of replicative senescence, a cellular state where cells enter an irreversible growth arrest. Induction of p21 is required to initiate the process, and to inhibit cdk2-cyclin E complexes. The 5′ UTR of p21 contains a GC-rich sequence that forms a stem loop. This element is recognized by two RBPs with distinct properties: CUGBP1 and calreticulin (CRT). Competition between the two proteins determines final levels of p21 expression and establishes if cells will proliferate or undergo growth arrest and senescence. refers to: Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate, P. Iakova, G. L. Wang, L. Timchenko et al EMBO J 23(2), 406-417, 2004.

IRP simultaneously affects mRNA translation and stability

Müllner et al. (1989). A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell 58, 373-382.

rare case: degradation via a specific endonuclease!

consensus structure of an IRE

Abundance of factors binding to regulatory sites in mRNA affects outcome

Asymmetric mRNA distribution in differentiated cells

Left: ß-actin mRNA localized to the leading process in chicken fibroblasts. ß-actin mRNA in red, actin protein green, nuclei in blue.

30 sec movie on cell movement in general: http://www.youtube.com/watch?v=vAaQ4WS8QhI&NR=1 Right: c-myc mRNA staining in a fibroblast cell, showing perinuclear accumulation of the mRNA.

Ralf-Peter Jansen, mRNA localization: Message on the move. Nat Revs Mol Cell Biol 2, 247-256, (2001)


Mechanisms of ‘normal’ mRNA degradation

PARN = polyA-dependent ribonuclease

The highways and byways of mRNA decay Nicole L. Garneau, Jeffrey Wilusz and Carol J. Wilusz Nature Reviews Molecular Cell Biology 8, 113-126 (February 2007)

a | deadenylation-dependent b | deadenylation-independent (yeast)

c | endonuclease-mediated

Abbreviations: UTR, untranslated region; m7G, 7-methyl-guanosine cap; hairpin, hairpin-like secondary structures; uORF, upstream open reading frame; IRES, internal ribosome entry site; CPE, cytoplasmic polyadenylation element; AAUAAA, polyadenylation signal.

Mignone et al. Genome Biology 2002 3:reviews0004.1 doi:10.1186/gb-2002-3-3-reviews0004

AUUUA

stability

Post-transcriptional regulatory elements affecting gene expression almost a summary of stuff already covered and out-look for SS2015

Post-transcriptional regulatory elements affecting gene expression

sometimes, the older figure is the better one but look to the title of the review, next slide

end of regular part

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