IRES (biology)

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As internal ribosomal entry site ( English internal ribosomal entry site ), abbreviated IRES is in cell biology a specific folded portion within an RNA referred -Einzelstrangs, the binding to ribosomes conveyed. The secondary structure of an IRES makes it possible to initiate the translation in the synthesis of proteins in eukaryotes independently of the 5'-cap structure , for example also from the middle of a messenger RNA (mRNA). IRES have so far been found in the RNA of viruses and described in the mRNA for some genes of cells .

history

In 1988 an internal ribosomal entry site (IRES) in the RNA genome of the poliovirus and the encephalomyocarditis virus was described for the first time in the laboratories of Nahum Sonenberg and Eckard Wimmer . The process of the translation start mediated by this was referred to as internal initiation of translation .

Basics

Usually, eukaryotic cellular mRNAs have a specially attached nucleotide at their 5 'end, the so-called 5' cap structure , which, together with other cellular factors, mediates the binding to ribosomes . In protein biosynthesis , this usually initiates translation. Some viral genomes are excluded from this complex control system, which can bind directly to the 40S subunit of ribosomes by means of an IRES and thus induce the synthesis of proteins typical of viruses by the cellular synthesis apparatus. In addition, for some of their genes in eukaryotes - for example for the oncogene c-Myc , the ornithine decarboxylase , the fibroblast growth factor 2 or the endothelial growth factor - special secondary structures have been described in the transcribed mRNAs that act in a similar way as internal ribosomal entry points of protein synthesis work. A further alternative mechanism for initiating translation is also possible via cap-independent translation elements.

Some of the IRES are linked to disease; the loss of function of an IRES element on the mRNA of the connexin 32 gene seems to be a main cause of Charcot-Marie-Tooth disease .

The molecular mechanisms of viral IRES are more extensively characterized than those of the eukaryotic ones. The IRES of the hepatitis C virus binds directly to the P-site of the 40S ribosomal subunit, so no eukaryotic initiation factors such as eIF1, 1A, 4A, 4B and 4E are necessary with this IRES. The picornavirus -IRES binds to the 40S subunit via eIF4. Some viral and eukaryotic IRES require additional cellular proteins called IRES-trans-acting factors (ITAF).

Viral IRES

Secondary structures of the IRES in the RNA genome of the poliovirus

Some RNA viruses have an IRES, with which the production of viral proteins on ribosomes of the cell can be started independently of initiation factors. This applies, for example, to the hepatitis C virus and the picornaviruses , e.g. B. the poliovirus . The IRES structure was discovered in the poliovirus, which blocks the synthesis of cellular proteins in cell culture in favor of the viral products. Since the cellular initiation factor eIF4G is cleaved by viral enzymes, only those RNA strands that contain an IRES like the virus RNA can be translated. Variants of an IRES that differ from this occur in Dicistroviridae .

Examples of viral and cellular IRES

IRES in viral genomes
virus IRES
Poliovirus Picornavirus IRES
Rhinovirus Picornavirus IRES
Encephalomyocarditis virus Picornavirus IRES
Foot-and-mouth disease virus Aphthovirus IRES
Hepatitis A virus Hepatitis A IRES
Hepatitis C virus Hepatitis C IRES
Classical swine fever virus Pestivirus IRES
Bovine viral diarrhea virus Pestivirus IRES
Friend Murine Leukemia Virus
Moloney Murines Leukemia Virus (MMLV)
Rous sarcoma virus
Human immunodeficiency virus
Plautia stali intestine virus Cripavirus internal ribosome entry site (IRES)
Rhopalosiphum padi virus Cripavirus internal ribosome entry site (IRES)
Cricket paralysis virus Cripavirus internal ribosome entry site (IRES)
Triatoma virus Cripavirus internal ribosome entry site (IRES)
Kaposi's sarcoma-associated herpesvirus Kaposi's sarcoma-associated herpesvirus IRES
Marek's disease virus MDV 5'Leader IRES and intercistronic IRES in the 1.8-kb family of immediate early transcripts (IRES) 1
IRES in cellular mRNA
Protein type Proteins
Growth factors Fibroblast growth factor ( FGF-1 IRES and 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) a, Eukaryotic initiation factor 4Gl (elF4Gl) a, Death associated protein 5 (DAP5)
Oncogenes c-myc , L-myc , Pim-1, protein kinase p58PITSLRE, p53
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 in neural 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
Other Immunoglobulin heavy chain binding protein (BiP) , Heat shock protein 70 , β-subunit of mitochondrial H + -ATP synthase, Ornithine decarboxylase , connexins 32 and 43 , HIF-1α , APC

Applications

IRES elements are also used in the course of a vector design , for example for the coexpression of a reporter gene to control the transfection or transduction efficiency . The gene to be cloned is usually placed in front of the IRES (in the 5 'direction), the reporter gene behind it. The expression of the reporter gene indicates the synthesis of the full length mRNA.

Limitations and Alternatives

Viral IRES sequences are widely used in technical molecular biology to mimic polycistronic mRNA. For this purpose, several genes are cloned one after the other on a plasmid and an IRES sequence is inserted between the genes. With this construct, only a single promoter and terminator is required. However, the introduced IRES sequences have the disadvantage that the expression efficiency for each subsequent gene is reduced.

The viral 2A peptides represent a technical alternative to the IRES . When using 2A peptide sequences, the gene regulation of a bacterial operon is imitated and an artificially created sequence of several genes in a common open reading frame is regulated via just one promoter. The self-cleaving 2A peptides - oligopeptides made up of around twenty amino acids - are used to break down the mutually expressed gene products into individual proteins. The advantage here compared to using IRES sequences is the equimolar expression of the proteins via the common promoter. However, after cleavage of the 2A peptides, short peptide portions remain at the termini of the expressed proteins, which can disrupt their function.

Individual evidence

  1. ^ SR Thompson: Tricks an IRES uses to enslave ribosomes. In: Trends in microbiology. Volume 20, number 11, November 2012, pp. 558-566, ISSN  1878-4380 , doi: 10.1016 / j.tim.2012.08.002 , PMID 22944245 , PMC 3479354 (free full text).
  2. ↑ Pelletier J, Sonenberg N: Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA . In: Nature . 334, No. 6180, 1988, pp. 320-5. doi : 10.1038 / 334320a0 . PMID 2839775 .
  3. ↑ Jang SK, Kräusslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E: A segment of the 5 'nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation . In: J. Virol. . 62, No. 8, August 1988, pp. 2636-43. PMID 2839690 . PMC 253694 (free full text).
  4. ^ AA Komar, B. Mazumder, WC Merrick: A new framework for understanding IRES-mediated translation. In: Genes. Volume 502, Number 2, July 2012, pp. 75-86, ISSN  1879-0038 . doi: 10.1016 / j.gene.2012.04.039 . PMID 22555019 . PMC 3361623 (free full text).
  5. ↑ A. Huddler, R. Werner: Analysis of a Charcot-Marie-Tooth Disease Mutation Reveals an Essential Internal Ribosome Entry Site Element in the Connexin-32 Gene .
  6. ↑ López-Lastra M, Rivas A, Barría MI: Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation . In: Biol. Res. . 38, No. 2-3, 2005, pp. 121-46. PMID 16238092 .
  7. ↑ a b c Hellen CU, Sarnow P: Internal ribosome entry sites in eukaryotic mRNA molecules . In: Genes Dev. . 15, No. 13, 2001, pp. 1593-612. doi : 10.1101 / gad.891101 . PMID 11445534 .
  8. ^ E. Jan: Divergent IRES elements in invertebrates. In: Virus research. Volume 119, Number 1, July 2006, pp. 16-28, ISSN  0168-1702 . doi: 10.1016 / j.virusres.2005.10.011 . PMID 16307820 .
  9. ↑ Kozak M: A second look at cellular mRNA sequences said to function as internal ribosome entry sites . In: Nucleic Acids Res . 33, No. 20, 2005, pp. 6593-602. doi : 10.1093 / nar / gki958 . PMID 16314320 . PMC 1298923 (free full text).
  10. ↑ Donna Michnick, Louise C. Wasley, Monique V. Davies, Randal J. Kaufman: Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus . In: Nucleic Acids Research . tape 19 , no. 16 , 25 August 1991, ISSN  0305-1048 , pp. 4485-4490 , doi : 10.1093 / nar / 19.16.4485 .
  11. ↑ Qingyou Xia, Ping Zhao, Riyuan Wang, Feng Wang, Yuancheng Wang: 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori . In: Scientific Reports . tape 5 , November 5, 2015, p. 16273 , doi : 10.1038 / srep16273 , PMID 26537835 , PMC 4633692 (free full text).
  12. ↑ AL Szymczak-Workman, KM Vignali, DAA Vignali: Design and Construction of 2A Peptide-Linked Multicistronic Vectors . In: Cold Spring Harbor Protocols . tape 2012 , no. February 2 , 2012, ISSN  1559-6095 , doi : 10.1101 / pdb.ip067876 .

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