Cap-Independent Translation: What’s in a Name? Shatsky, Ivan N.; Terenin, Ilya M.; Smirnova, Victoria V. ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
November 2018, 2018-11-00, 20181101, Volume:
43, Issue:
11
Journal Article
Peer reviewed
Eukaryotic translation initiation relies on the m7G cap present at the 5′ end of all mRNAs. Some viral mRNAs employ alternative mechanisms of initiation based on internal ribosome entry. The ‘IRES ...ideology’ was adopted by researchers to explain the differential translation of cellular mRNAs when the cap recognition is suppressed. However, some cellular IRESs have already been challenged and others are awaiting their validation. As an alternative cap-independent mechanism, we propose adopting the concept of cap-independent translation enhancers (CITEs) for mammalian mRNAs. Unlike IRESs, CITEs can be located both within 5′ and 3′ UTRs and bind mRNA-recruiting translational components. The respective 5′ UTRs are then inspected by the scanning machinery essentially in the same way as under cap-dependent translation.
The eukaryotic translation initiation relies not only on the cap-dependent mechanism of mRNA binding to the ribosome, but also on poorly studied cap-independent ways of recruiting mRNAs to ribosomes. The latter mode plays an important role in cell differentiation and cellular response to abnormal conditions.
The widely adopted way to explain the cap-independent translation of cellular mRNAs is the use of internal ribosome entry sites (IRESs). However, some of the cap-independent mechanisms cannot be explained with the IRES concept.
This review proposes an alternative mechanism for cap-independent initiation. It is based on the presence (in the UTRs of mRNAs) of structural elements that bind the factors recruiting mRNAs to the ribosome cap-independent translational enhancers (CITEs). This mechanism is supported by recent reports.
By binding components of the translation machinery, CITEs help specific mRNAs to win the competition for ribosomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel ...GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNAiMet occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ...ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
4.
A researcher’s guide to the galaxy of IRESs Terenin, Ilya M.; Smirnova, Victoria V.; Andreev, Dmitri E. ...
Cellular and molecular life sciences : CMLS,
04/2017, Volume:
74, Issue:
8
Journal Article
Peer reviewed
The idea of internal initiation is frequently exploited to explain the peculiar translation properties or unusual features of some eukaryotic mRNAs. In this review, we summarize the methods and ...arguments most commonly used to address cases of translation governed by internal ribosome entry sites (IRESs). Frequent mistakes are revealed. We explain why “cap-independent” does not readily mean “IRES-dependent” and why the presence of a long and highly structured 5′ untranslated region (5′UTR) or translation under stress conditions cannot be regarded as an argument for appealing to internal initiation. We carefully describe the known pitfalls and limitations of the bicistronic assay and artefacts of some commercially available in vitro translation systems. We explain why plasmid DNA transfection should not be used in IRES studies and which control experiments are unavoidable if someone decides to use it anyway. Finally, we propose a workflow for the validation of IRES activity, including fast and simple experiments based on a single genetic construct with a sequence of interest.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and ...favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known.
We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days.
Established viral replication does not cause global changes in host gene expression-only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (
/
,
/
, and
) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes.
After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming ("Warburg effect") even in the hepatocellular carcinoma cells used here.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are ...directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3'-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in
, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and ...replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.
Abstract
The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the ...recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5′ untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5′ ends without the involvement of either the 5′ m7G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.
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BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
► Translation of some mRNAs under stress conditions is thought to involve IRESs. ► The 5′ UTR of human Apaf-1 mRNA can direct cap-independent initiation during apoptosis. ► However, the Apaf-1 5′ UTR ...does not use IRES under apoptosis. ► The underlying mechanism remains a 5′end-dependent initiation followed by scanning.
We have previously shown that translation driven by the 5′ UTR of Apaf-1 mRNA is relatively efficient in the absence of m7G-cap, but no IRES is involved. Nevertheless, it may be speculated that a “silent” IRES is activated under apoptosis conditions. Here, we show that translation of the mRNA with the Apaf-1 5′ UTR is relatively resistant to apoptosis induced by etoposide when eIF4E is sequestered by 4E-BP and eIF4G is partially cleaved. However, translation under these conditions remains governed by 5′ end-dependent scanning. We hypothesize that the observed phenomenon is based on the intrinsic low cap-dependence of the Apaf-1 5′ UTR.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eukaryotic cells evolved highly complex and accurate protein synthesis machinery that is finely tuned by various signaling pathways. Dysregulation of translation is a hallmark of many diseases, ...including cancer, and thus pharmacological approaches to modulate translation become very promising. While there has been much progress in our understanding of mammalian mRNA-specific translation control, surprisingly, relatively little is known about whether and how the protein components of the translation machinery shape translation of their own mRNAs. Here we analyze mammalian mRNAs encoding components of the translation initiation machinery for potential regulatory features such as 5′TOP motifs, TISU motifs, poor start codon nucleotide context and upstream open reading frames.
•mRNAs encoding translation initiation factors (eIFs) rarely have 5’TOP motifs•Instead, translation of some eIF mRNAs is regulated by start codon selection (either of uORFs or CDS)•eIF4B mRNA translation is likely controlled through a TISU motif•For multisubunit eIFs, mRNAs encoding different subunits have varying features possibly implicated in translation regulation
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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