Статті в журналах: "Ribosomal leaky scanning"

1

Smirnova, Victoria V., Ekaterina D. Shestakova, Daria S. Nogina, Polina A. Mishchenko, Tatiana A. Prikazchikova, Timofei S. Zatsepin, Ivan V. Kulakovskiy, Ivan N. Shatsky, and Ilya M. Terenin. "Ribosomal leaky scanning through a translated uORF requires eIF4G2." Nucleic Acids Research 50, no. 2 (January 8, 2022): 1111–27. http://dx.doi.org/10.1093/nar/gkab1286.

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Abstract eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5′ UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5′ UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.

2

Zhou, Weihui, and Weihong Song. "Leaky Scanning and Reinitiation Regulate BACE1 Gene Expression." Molecular and Cellular Biology 26, no. 9 (May 1, 2006): 3353–64. http://dx.doi.org/10.1128/mcb.26.9.3353-3364.2006.

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ABSTRACT β-Site β-amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is the β-secretase in vivo for processing APP to generate amyloid β protein (Aβ). Aβ deposition in the brain is the hallmark of Alzheimer's disease (AD) neuropathology. Inhibition of BACE1 activity has major pharmaceutical potential for AD treatment. The expression of the BACE1 gene is relatively low in vivo. The control of BACE1 expression has not been well defined. There are six upstream AUGs (uAUGs) in the 5′ leader sequence of the human BACE1 mRNA. We investigated the role of the promoter and the uATGs in the 5′ untranslated region (UTR) of the human BACE1 gene in BACE1 gene transcription and translation initiation. Our results show that the first and second uATGs are the integral part of the core minimal promoter of the human BACE1 gene, while the third uAUG is skipped over by ribosomal scanning. The fourth uAUG can function as a translation initiation codon, and deletion or mutation of this uAUG increases downstream gene expression. The fourth uAUG of the BACE1 5′UTR is responsible for inhibiting the expression of BACE1. Translation initiation by the BACE1 uAUGs and physiological AUG requires intact eIF4G. Our results demonstrate that during human BACE1 gene expression, ribosomes skipped some uAUGs by leaky scanning and translated an upstream open reading frame, initiated efficiently at the fourth uAUG, and subsequently reinitiated BACE1 translation at the physiological AUG site. Such leaky scanning and reinitiation resulted in weak expression of BACE1 under normal conditions. Alterations of the leaky scanning and reinitiation in BACE1 gene expression could play an important role in AD pathogenesis.

3

Pisareva, Vera P., and Andrey V. Pisarev. "DHX29 reduces leaky scanning through an upstream AUG codon regardless of its nucleotide context." Nucleic Acids Research 44, no. 9 (April 11, 2016): 4252–65. http://dx.doi.org/10.1093/nar/gkw240.

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Abstract During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2α in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.

4

Schneider, P. A., R. Kim, and W. I. Lipkin. "Evidence for translation of the Borna disease virus G protein by leaky ribosomal scanning and ribosomal reinitiation." Journal of virology 71, no. 7 (1997): 5614–19. http://dx.doi.org/10.1128/jvi.71.7.5614-5619.1997.

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5

de Breyne, Sylvain, and Théophile Ohlmann. "Focus on Translation Initiation of the HIV-1 mRNAs." International Journal of Molecular Sciences 20, no. 1 (December 28, 2018): 101. http://dx.doi.org/10.3390/ijms20010101.

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To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins.

6

Liu, Q., and G. Hobom. "Evidence for translation of VP3 of avian polyomavirus BFDV by leaky ribosomal scanning." Archives of Virology 145, no. 2 (February 15, 2000): 407–16. http://dx.doi.org/10.1007/s007050050032.

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7

Smith, E. "Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo." Nucleic Acids Research 33, no. 4 (February 23, 2005): 1298–308. http://dx.doi.org/10.1093/nar/gki248.

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8

Ferreira, Joshua P., William L. Noderer, Alexander J. Diaz de Arce, and Clifford L. Wang. "Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation." Bioengineered 5, no. 3 (January 14, 2014): 186–92. http://dx.doi.org/10.4161/bioe.27607.

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9

Stacey, Simon N., Deborah Jordan, Andrew J. K. Williamson, Michael Brown, Joanna H. Coote, and John R. Arrand. "Leaky Scanning Is the Predominant Mechanism for Translation of Human Papillomavirus Type 16 E7 Oncoprotein from E6/E7 Bicistronic mRNA." Journal of Virology 74, no. 16 (August 15, 2000): 7284–97. http://dx.doi.org/10.1128/jvi.74.16.7284-7297.2000.

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ABSTRACT Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5′ untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopusβ-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5′ end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5′ end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

10

Latorre, Patrizia, Daniel Kolakofsky, and Joseph Curran. "Sendai Virus Y Proteins Are Initiated by a Ribosomal Shunt." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 5021–31. http://dx.doi.org/10.1128/mcb.18.9.5021.

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ABSTRACT The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C′, C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C′ to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5′ cap and then scan ca. 50 nucleotides (nt) of the 5′ untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

11

Firth, Andrew E., and Ian Brierley. "Non-canonical translation in RNA viruses." Journal of General Virology 93, no. 7 (July 1, 2012): 1385–409. http://dx.doi.org/10.1099/vir.0.042499-0.

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Viral protein synthesis is completely dependent upon the translational machinery of the host cell. However, many RNA virus transcripts have marked structural differences from cellular mRNAs that preclude canonical translation initiation, such as the absence of a 5′ cap structure or the presence of highly structured 5′UTRs containing replication and/or packaging signals. Furthermore, whilst the great majority of cellular mRNAs are apparently monocistronic, RNA viruses must often express multiple proteins from their mRNAs. In addition, RNA viruses have very compact genomes and are under intense selective pressure to optimize usage of the available sequence space. Together, these features have driven the evolution of a plethora of non-canonical translational mechanisms in RNA viruses that help them to meet these challenges. Here, we review the mechanisms utilized by RNA viruses of eukaryotes, focusing on internal ribosome entry, leaky scanning, non-AUG initiation, ribosome shunting, reinitiation, ribosomal frameshifting and stop-codon readthrough. The review will highlight recently discovered examples of unusual translational strategies, besides revisiting some classical cases.

12

Kirshner, Jessica R., Katherine Staskus, Ashley Haase, Michael Lagunoff, and Don Ganem. "Expression of the Open Reading Frame 74 (G-Protein-Coupled Receptor) Gene of Kaposi’s Sarcoma (KS)-Associated Herpesvirus: Implications for KS Pathogenesis." Journal of Virology 73, no. 7 (July 1, 1999): 6006–14. http://dx.doi.org/10.1128/jvi.73.7.6006-6014.1999.

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ABSTRACT Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) encodes a G-protein-coupled receptor (GCR) homolog. This protein is a potent, constitutively active signalling molecule that can influence both proliferation and angiogenesis when ectopically expressed in fibroblasts in vitro. Here we have examined the expression of the KSHV GCR gene in virus-infected lymphoid cells and in KS tumors. Our results show that in both situations the gene is expressed primarily during lytic replication; its transcription is unaffected by inhibition of viral DNA synthesis, indicating that it is expressed in the early phases of the lytic program. The major transcript bearing GCR sequences is bicistronic, harboring coding sequences for another viral gene, K14, at its 5′ end. Extensive searches for monocistronic GCR mRNAs using nuclease mapping and reverse transcription-PCR failed to detect such species. The 5′ end of K14/GCR mRNA maps to nucleotide (nt) 127848, and its poly(A) addition site maps to nt 130546; a 149-nt intron is present in the K14/GCR intergenic region. These results suggest that the KSHV GCR is translated by unconventional mechanisms involving either translational reinitiation, internal ribosomal entry, or leaky ribosomal scanning. The restriction of GCR expression to the lytic cycle has important implications for the potential role(s) of the GCR in KS pathogenesis.

13

Levine, Corinna G., Devarati Mitra, Ajay Sharma, Carolyn L. Smith, and Ramanujan S. Hegde. "The Efficiency of Protein Compartmentalization into the Secretory Pathway." Molecular Biology of the Cell 16, no. 1 (January 2005): 279–91. http://dx.doi.org/10.1091/mbc.e04-06-0508.

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Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins.

14

Chenik, M., K. Chebli, and D. Blondel. "Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism." Journal of virology 69, no. 2 (1995): 707–12. http://dx.doi.org/10.1128/jvi.69.2.707-712.1995.

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15

Chatterji, Udayan, Aymeric de Parseval, and John H. Elder. "Feline Immunodeficiency Virus OrfA Is Distinct from Other Lentivirus Transactivators." Journal of Virology 76, no. 19 (October 1, 2002): 9624–34. http://dx.doi.org/10.1128/jvi.76.19.9624-9634.2002.

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ABSTRACT The feline immunodeficiency virus (FIV) accessory factor, OrfA, facilitates transactivation of transcription directed by elements of the viral long terminal repeat (LTR). In order to map OrfA domains required for this transactivation, we used N- and C-terminal deletion constructs of the protein, expressed in a Gal4-based transactivation system. The results demonstrated that FIV OrfA, unlike other lentiviral transactivators such as visna virus Tat, is unable to transactivate from minimal promoter-based reporters and requires additional elements of the viral LTR. Stable CrFK-based cell lines were prepared that expressed OrfA to readily detectable levels and in which we were able to demonstrate 32-fold transactivation of an LTR-chloramphenicol acetyltransferase construct. Transactivation was heavily dependent on the presence of an ATF site within the viral LTR. Changing the translation initiation codon context substantially increased the level of production of OrfA from a bicistronic message that also encodes Rev. In the presence of a more favorable context sequence, the upstream expression of OrfA increased 21-fold, with only a 0.5-fold drop in downstream Rev expression. This suggests that Rev translation may occur via an internal ribosomal entry site rather than by leaky scanning.

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Lin, Ching-Gong, and Szecheng J. Lo. "Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus Pol gene from the viral pregenome RNA." Virology 188, no. 1 (May 1992): 342–52. http://dx.doi.org/10.1016/0042-6822(92)90763-f.

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17

Singh, Chingakham Ranjit, Cynthia Curtis, Yasufumi Yamamoto, Nathan S. Hall, Dustin S. Kruse, Hui He, Ernest M. Hannig, and Katsura Asano. "Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5480–91. http://dx.doi.org/10.1128/mcb.25.13.5480-5491.2005.

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ABSTRACT The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAi Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.

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Hinton, Tracey M., Feng Li, and Brendan S. Crabb. "Internal Ribosomal Entry Site-Mediated Translation Initiation in Equine Rhinitis A Virus: Similarities to and Differences from That of Foot-and-Mouth Disease Virus." Journal of Virology 74, no. 24 (December 15, 2000): 11708–16. http://dx.doi.org/10.1128/jvi.74.24.11708-11716.2000.

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ABSTRACT Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5′ nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.

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Kumar, Amod, Asaf V. N. Muhasin, Ashwin Ashok Raut, Richa Sood, and Anamika Mishra. "Identification of Chicken Pulmonary miRNAs Targeting PB1, PB1-F2, and N40 Genes of Highly Pathogenic Avian Influenza Virus H5N1 in Silico." Bioinformatics and Biology Insights 8 (January 2014): BBI.S14631. http://dx.doi.org/10.4137/bbi.s14631.

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Highly pathogenic Avian influenza (HPAI) is a notifiable viral disease caused by avian influenza type A viruses of the Orthomyxoviridae family. Type A influenza genome consists of eight segments of negative-sense RNA. RNA segment 2 encodes three proteins, PB1, PB1-F2, and N40, which are translated from the same mRNA by ribosomal leaky scanning and reinitiation. Since these proteins are critical for viral replication and pathogenesis, targeting their expression can be one of the approaches to control and resist HPAI. MicroRNAs are short noncoding RNAs that regulate a variety of biological processes such as cell growth, tissue differentiation, apoptosis, and viral infection. In this study, a set of 300 miRNAs expressed in chicken lungs were screened against the HPAI virus (H5N1) segment 2 with different screening parameter like thermodynamic stability of heteroduplex, seed sequence complementarity, conserved target sequence, and target-site accessibility for identifying miRNAs that can potentially target the transcript of segment 2 of H5N1. Chicken miRNAs gga-mir-133c, gga-mir-1710, and gga-mir-146c* are predicted to target the expression of PB1, PB1-F2, and N40 proteins. This indicates that chicken has genetic potential to resist/tolerate H5N1 infection and these can be suitably exploited in designing strategies for control of avian influenza in chicken.

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Hsieh, Ching-Chyuan, Wei Xiong, Qizhi Xie, Jeffrey P. Rabek, Sheen G. Scott, Mi Ra An, Peter D. Reisner, David T. Kuninger та John Papaconstantinou. "Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers". Molecular Biology of the Cell 9, № 6 (червень 1998): 1479–94. http://dx.doi.org/10.1091/mbc.9.6.1479.

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The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPαand p30C/EBPαin control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPαlevels and binding activity, whereas those of p20C/EBPαand p20C/EBPβare increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

21

Tauber, Sandra, Yvonne Ligertwood, Marlynne Quigg-Nicol, Bernadette M. Dutia, and Richard M. Elliott. "Behaviour of influenza A viruses differentially expressing segment 2 gene products in vitro and in vivo." Journal of General Virology 93, no. 4 (April 1, 2012): 840–49. http://dx.doi.org/10.1099/vir.0.039966-0.

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The influenza A virus genome comprises eight segments of negative-sense RNA that encode up to 12 proteins. RNA segment 2 encodes three proteins, PB1, PB1-F2 and N40, that are translated from the same mRNA by ribosomal leaky scanning and reinitiation. PB1 is a subunit of the trimeric viral RNA polymerase. PB1-F2 has been reported to be a potential virulence factor, and has been shown to be involved in a number of activities including induction of apoptosis, regulation of virus replication and modulation of the immune response. No function has yet been ascribed to N40, which represents an N-terminally deleted form of PB1. Previous studies on PB1-F2 function mainly used viruses genetically engineered to prevent PB1-F2 expression by mutation of the PB1-F2 start codon. However, ablation of the start codon was shown to increase the expression level of the downstream protein N40. In the present study, we generated recombinant A/WSN/33 viruses carrying different combinations of PB1-F2- and N40-knockout mutations. Overexpression of N40 in a PB1-F2-deficient background had a detrimental effect on virus growth in vitro and in vivo. However, ablation of PB1-F2 or N40 expression individually was not disadvantageous for the virus. Primer-extension analyses revealed an increase in vRNA production by viruses that overexpressed N40. Our data suggest that the observed attenuation of mutant viruses in vitro and in vivo results from these changes in transcription and replication.

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Hogan, Michael J., Nikita Maheshwari, Annalisa Nicastri, Nicola Ternette, and Laurence C. Eisenlohr. "Immunodominant, Qa-1-restricted CD8 T cell response elicited against a non-canonical translation product in influenza A virus." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 103.05. http://dx.doi.org/10.4049/jimmunol.206.supp.103.05.

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Abstract The extent to which non-canonical translation products drive antiviral T cell responses is incompletely understood. We explored this question using an immunopeptidomic approach in an influenza virus model. C57Bl/6 mouse-derived cells were infected with influenza virus A/Puerto Rico/8/1934 (PR8), and MHC-bound peptides were eluted and analyzed by tandem mass spectrometry. Mass spectra were searched against a database of all viral gene segments translated in all possible reading frames, irrespective of the presence of start codons. We identified a 9-mer peptide, here termed M-SL9, that maps to an alternative reading frame of the sequence encoding matrix protein 1 (M1). To validate M-SL9 as a T cell epitope, we infected C57Bl/6 mice with PR8, collected tissues, and measured T cell reactivation by synthetic M-SL9 peptide. Remarkably, 10% of all CD8 T cells in the lung were specifically reactivated by M-SL9 peptide, and the vast majority of these were polyfunctional, producing two or more Th1 cytokines. An analysis of M-SL9 processing and presentation revealed that M-SL9 is restricted to the non-classical MHC class Ib molecule, Qa-1, and that presentation is dependent on the second 5′-proximal AUG codon within the M1-coding sequence, suggesting that its translation results from leaky ribosomal scanning. These data contribute to a growing body of work showing the importance of non-canonical MHC presentation in protective immune responses.

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Shestakova, Ekaterina D., Roman S. Tumbinsky, Dmitri E. Andreev, Fedor N. Rozov, Ivan N. Shatsky, and Ilya M. Terenin. "The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA." International Journal of Molecular Sciences 24, no. 24 (December 5, 2023): 17149. http://dx.doi.org/10.3390/ijms242417149.

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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.

24

Matsuda, Daiki, Lisa Bauer, Kathryn Tinnesand, and Theo W. Dreher. "Expression of the Two Nested Overlapping Reading Frames of Turnip Yellow Mosaic Virus RNA Is Enhanced by a 5′ Cap and by 5′ and 3′ Viral Sequences." Journal of Virology 78, no. 17 (September 1, 2004): 9325–35. http://dx.doi.org/10.1128/jvi.78.17.9325-9335.2004.

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ABSTRACT The translation efficiency of an mRNA molecule is typically determined by its 5′- and/or 3′-untranslated regions (UTRs). Previously, we have found that the 3′-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5′ cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5′ 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5′-UTR. Maximum expression was observed from RNAs with a cap and both 5′ and 3′ TYMV sequences. In paired reporter constructs, the 5′ 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG69 and AUG206) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5′ cap, with a threefold-higher expression occurring from AUG69 than from AUG206 in the presence of the genomic 3′-UTR. Changes that interrupted the cap/3′-UTR synergy (i.e., removal of the cap or TYMV 3′-UTR) resulted in a higher proportion of initiation from AUG206. Mutation of the 3′-UTR to prevent aminoacylation, as well as deletion of 75% of the 5′-UTR, likewise resulted in a lower ratio of expression from AUG69 relative to AUG206. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG69 and AUG206. However, our observations do not support a recent proposal based on in vitro studies in which the 3′-UTR is proposed to direct cap-independent initiation specifically at AUG206 and not at AUG69 (S. Barends et al., Cell 112:123-129, 2003).

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Firth, Andrew E., Jessika C. Zevenhoven-Dobbe, Norma M. Wills, Yun Young Go, Udeni B. R. Balasuriya, John F. Atkins, Eric J. Snijder, and Clara C. Posthuma. "Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production." Journal of General Virology 92, no. 5 (May 1, 2011): 1097–106. http://dx.doi.org/10.1099/vir.0.029264-0.

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The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3′-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5′ end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43–64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection.

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Andreev, D. E., P. V. Baranov, A. Milogorodskii, and D. Rachinskii. "A deterministic model for non-monotone relationship between translation of upstream and downstream open reading frames." Mathematical Medicine and Biology: A Journal of the IMA 38, no. 4 (October 29, 2021): 490–515. http://dx.doi.org/10.1093/imammb/dqab015.

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Abstract Totally asymmetric simple exclusion process (TASEP) modelling was shown to offer a parsimonious explanation for the experimentally confirmed ability of a single upstream open reading frames (uORFs) to upregulate downstream translation during the integrated stress response. As revealed by numerical simulations, the model predicts that reducing the density of scanning ribosomes upstream of certain uORFs increases the flow of ribosomes downstream. To gain a better insight into the mechanism which ensures the non-monotone relation between the upstream and downstream flows, in this work, we propose a phenomenological deterministic model approximating the TASEP model of the translation process. We establish the existence of a stationary solution featuring the decreasing density along the uORF for the deterministic model. Further, we find an explicit non-monotone relation between the upstream ribosome density and the downstream flow for the stationary solution in the limit of increasing uORF length and increasingly leaky initiation. The stationary distribution of the TASEP model, the stationary solution of the deterministic model and the explicit limit are compared numerically.

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Racine, Trina, Chris Barry, Kenneth Roy, Sandra J. Dawe, Maya Shmulevitz, and Roy Duncan. "Leaky Scanning and Scanning-independent Ribosome Migration on the Tricistronic S1 mRNA of Avian Reovirus." Journal of Biological Chemistry 282, no. 35 (June 29, 2007): 25613–22. http://dx.doi.org/10.1074/jbc.m703708200.

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28

Sun, Tony, Yingpu Yu, Xianfang Wu, Ashley Acevedo, Ji-Dung Luo, Jiayi Wang, William M. Schneider, et al. "Decoupling expression and editing preferences of ADAR1 p150 and p110 isoforms." Proceedings of the National Academy of Sciences 118, no. 12 (March 15, 2021): e2021757118. http://dx.doi.org/10.1073/pnas.2021757118.

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Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.

29

Schwartz, S., B. K. Felber, and G. N. Pavlakis. "Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs." Molecular and Cellular Biology 12, no. 1 (January 1992): 207–19. http://dx.doi.org/10.1128/mcb.12.1.207-219.1992.

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We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.

30

Schwartz, S., B. K. Felber, and G. N. Pavlakis. "Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs." Molecular and Cellular Biology 12, no. 1 (January 1992): 207–19. http://dx.doi.org/10.1128/mcb.12.1.207.

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We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.

31

Verchot, Jeanmarie, Susan M. Angell, and David C. Baulcombe. "In Vivo Translation of the Triple Gene Block of Potato Virus X Requires Two Subgenomic mRNAs." Journal of Virology 72, no. 10 (October 1, 1998): 8316–20. http://dx.doi.org/10.1128/jvi.72.10.8316-8320.1998.

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ABSTRACT The 25-kilodalton (25K), 12K, and 8K movement proteins of potato virus X are derived from overlapping open reading frames (ORFs). Using an in vivo complementation assay, we have shown that the 25K protein is expressed from a functionally monocistronic mRNA, whereas the 12K and 8K proteins are from a bicistronic mRNA. Translation of the 8K ORF is by leaky ribosome scanning through the 12K ORF.

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Frederiks, Floor, Guus J. J. E. Heynen, Sjoerd J. van Deventer, Hans Janssen, and Fred van Leeuwen. "Two Dot1 isoforms in Saccharomyces cerevisiae as a result of leaky scanning by the ribosome." Nucleic Acids Research 37, no. 21 (September 24, 2009): 7047–58. http://dx.doi.org/10.1093/nar/gkp765.

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33

Wamboldt, Yashitola, Saleem Mohammed, Christian Elowsky, Chris Wittgren, Wilson B. M. de Paula, and Sally A. Mackenzie. "Participation of Leaky Ribosome Scanning in Protein Dual Targeting by Alternative Translation Initiation in Higher Plants." Plant Cell 21, no. 1 (January 2009): 157–67. http://dx.doi.org/10.1105/tpc.108.063644.

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34

Anderson, Jenny L., Adam T. Johnson, Jane L. Howard, and Damian F. J. Purcell. "Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs." Journal of Virology 81, no. 9 (February 28, 2007): 4664–76. http://dx.doi.org/10.1128/jvi.01028-06.

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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5′ untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5′ UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.

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Sears, R. C., and L. Sealy. "Multiple forms of C/EBP beta bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat." Molecular and Cellular Biology 14, no. 7 (July 1994): 4855–71. http://dx.doi.org/10.1128/mcb.14.7.4855-4871.1994.

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In this report we demonstrate that C/EBP beta is a major component of three EFII DNA binding complexes, EFIIa, EFIIb, and EFIIc, which we have previously shown to specifically recognize a C/EBP consensus binding site found in the EFII enhancer sequence from the Rous sarcoma virus long terminal repeat (R. C. Sears and L. Sealy, J. Virol. 66:6338-6352, 1992). Three different forms of C/EBP beta, p42, p35, and p20, can bind the EFII DNA sequence as homodimers, and dimerization experiments show that EFIIa is a homodimer of p20 C/EBP beta, EFIIb is primarily composed of a p20/p35 heterodimer with minor amounts of p20/p42 heterodimer and p35 homodimer, and EFIIc is composed of p20 and/or p35 heterodimerized with a novel 60-kDa protein. p20 C/EBP beta is likely equivalent to the internally initiated translation product of C/EBP beta, LIP (liver inhibitor protein), described by P. Descombes and U. Schibler (Cell 67:569-579, 1991). In contrast to the low level of LIP expressed in liver, postulated to occur because of leaky ribosome scanning, we found high levels of expression of p20 C/EBP beta in fibroblasts and lymphocytes. In murine fibroblasts, p20 C/EBP beta has an extended half-life, four times longer than those of p42 and p35 C/EBP beta, which could contribute to its abundant accumulation in this cell type, even though its synthesis by leaky ribosome scanning might be inefficient. Interestingly, overexpression of either the long or short form of C/EBP beta represses EFII-mediated transcription, suggesting that another unidentified EFII transactivator(s) exists, which may be dominantly inhibited by C/EBP beta proteins, and/or that transactivation by C/EBP beta proteins requires posttranslational modifications that were lacking in the transient overexpression experiments.

36

Sears, R. C., and L. Sealy. "Multiple forms of C/EBP beta bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat." Molecular and Cellular Biology 14, no. 7 (July 1994): 4855–71. http://dx.doi.org/10.1128/mcb.14.7.4855.

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In this report we demonstrate that C/EBP beta is a major component of three EFII DNA binding complexes, EFIIa, EFIIb, and EFIIc, which we have previously shown to specifically recognize a C/EBP consensus binding site found in the EFII enhancer sequence from the Rous sarcoma virus long terminal repeat (R. C. Sears and L. Sealy, J. Virol. 66:6338-6352, 1992). Three different forms of C/EBP beta, p42, p35, and p20, can bind the EFII DNA sequence as homodimers, and dimerization experiments show that EFIIa is a homodimer of p20 C/EBP beta, EFIIb is primarily composed of a p20/p35 heterodimer with minor amounts of p20/p42 heterodimer and p35 homodimer, and EFIIc is composed of p20 and/or p35 heterodimerized with a novel 60-kDa protein. p20 C/EBP beta is likely equivalent to the internally initiated translation product of C/EBP beta, LIP (liver inhibitor protein), described by P. Descombes and U. Schibler (Cell 67:569-579, 1991). In contrast to the low level of LIP expressed in liver, postulated to occur because of leaky ribosome scanning, we found high levels of expression of p20 C/EBP beta in fibroblasts and lymphocytes. In murine fibroblasts, p20 C/EBP beta has an extended half-life, four times longer than those of p42 and p35 C/EBP beta, which could contribute to its abundant accumulation in this cell type, even though its synthesis by leaky ribosome scanning might be inefficient. Interestingly, overexpression of either the long or short form of C/EBP beta represses EFII-mediated transcription, suggesting that another unidentified EFII transactivator(s) exists, which may be dominantly inhibited by C/EBP beta proteins, and/or that transactivation by C/EBP beta proteins requires posttranslational modifications that were lacking in the transient overexpression experiments.

37

Racine, T., and R. Duncan. "Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus." Nucleic Acids Research 38, no. 20 (July 7, 2010): 7260–72. http://dx.doi.org/10.1093/nar/gkq611.

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Carroll, R., and D. Derse. "Translation of equine infectious anemia virus bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation codon." Journal of Virology 67, no. 3 (1993): 1433–40. http://dx.doi.org/10.1128/jvi.67.3.1433-1440.1993.

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Schaecher, Scott R., Jason M. Mackenzie, and Andrew Pekosz. "The ORF7b Protein of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Is Expressed in Virus-Infected Cells and Incorporated into SARS-CoV Particles." Journal of Virology 81, no. 2 (November 1, 2006): 718–31. http://dx.doi.org/10.1128/jvi.01691-06.

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ABSTRACT Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two—open reading frame 3a (ORF3a) and ORF7a—have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a −1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.

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Zajakina, Anna, Tatyana Kozlovska, Ruta Bruvere, Jekaterina Aleksejeva, Paul Pumpens, and Henrik Garoff. "Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression." Journal of General Virology 85, no. 11 (November 1, 2004): 3343–51. http://dx.doi.org/10.1099/vir.0.80388-0.

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Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3′-truncated pgRNA lacking 3′ direct repeat and Pol mRNA, both carrying internally the HBs sequences, provided the synthesis of three HBs protein forms in the SFV-driven expression system. Maximal production of the HBs was provided by Pol mRNA, while HBc- and preC-producing templates showed relatively low internal translation of the HBs. These data allow the proposal of a ribosome leaky scanning model of internal translation initiation for HBs proteins. The putative functional role of such exceptional synthesis of the HBs proteins from the pgRNA and pcRNA templates in the natural HBV infection process needs further evaluation.

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Lin, Changyi A., Steven R. Ellis, and Heather L. True. "The Sua5 Protein Is Essential for Normal Translational Regulation in Yeast." Molecular and Cellular Biology 30, no. 1 (November 2, 2009): 354–63. http://dx.doi.org/10.1128/mcb.00754-09.

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ABSTRACT The anticodon stem-loop of tRNAs requires extensive posttranscriptional modifications in order to maintain structure and stabilize the codon-anticodon interaction. These modifications also play a role in accommodating wobble, allowing a limited pool of tRNAs to recognize degenerate codons. Of particular interest is the formation of a threonylcarbamoyl group on adenosine 37 (t6A37) of tRNAs that recognize ANN codons. Located adjacent and 3′ to the anticodon, t6A37 is a conserved modification that is critical for reading frame maintenance. Recently, the highly conserved YrdC/Sua5 family of proteins was shown to be required for the formation of t6A37. Sua5 was originally identified in a screen by virtue of its ability to affect expression from an aberrant upstream AUG codon in the cyc1 transcript. Together, these findings implicate Sua5 in protein translation at the level of codon recognition. Here, we show that Sua5 is critical for normal translation. The loss of SUA5 causes increased leaky scanning through AUG codons, +1 frameshifting, and nonsense suppression. In addition, the loss of SUA5 amplifies the 20S RNA virus found in Saccharomyces cerevisiae, possibly through an internal ribosome entry site-mediated mechanism. This study reveals a critical role for Sua5 and the t6A37 modification in translational fidelity.

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Wu, Yang, Zengpeng Han, Mingzhu Duan, Liangyu Jiang, Tiantian Tian, Dingyu Jin, Qitian Wang, and Fuqiang Xu. "Popularizing Recombinant Baculovirus-derived OneBac System for Laboratory Production of all Recombinant Adeno-associated Virus Vector Serotypes." Current Gene Therapy 21, no. 2 (April 19, 2021): 167–76. http://dx.doi.org/10.2174/1566523221666210118111657.

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Background: On the basis of our previously established single recombinant baculovirus expression vector (BEV)-derived OneBac system, we have optimized the process and expanded the rAAV production range to the full range of serotypes rAAV1-13. Objective: Recombinant adeno-associated virus (rAAV) has been widely used as an efficient transgenic vector in biomedical research, as well as gene therapy. Serotype-associated transduction efficiency, tissue- or cell-type tropism and immunological profile are major considerations in the various applications of rAAVs. There are increasing needs for different serotypes of rAAV, either naturally isolated or artificially engineered. However, affordable and scalable production of a desired serotype of rAAV remains very difficult, especially for researchers lacking relevant experience. Methods: Firstly, the AAV Cap gene was optimized to translate by ribosome leaky scanning and the gene of interest (GOI) was cloned into the pFD/Cap-(ITR-GOI)-Rep2 shuttle plasmid. Following the classical Bac-to-Bac method, sufficient BEV stock containing all rAAV packaging elements can be quickly obtained. Finally, we can repeatedly scale up the production of rAAVs in one week by using a single BEV to infect suspension-cultured Sf9 cells. The rAAV1-13 shows relatively high yields ranging from 5×104 to 4×105 VG/cell. More than 1×1015 VG purified rAAVs can be easily obtained from 5 L suspension-cultured Sf9 cells. Results: As expected, rAAV serotypes 1-13 show different potencies for in vitro transduction and cell-type tropisms. Conclusion: In summary, the single BEV-derived OneBac system should prove popular for laboratory scaling-up production of any serotype of rAAV.

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Haimov, Ora, Urmila Sehrawat, Ana Tamarkin-Ben Harush, Anat Bahat, Anna Uzonyi, Alexander Will, Hiroyuki Hiraishi, Katsura Asano, and Rivka Dikstein. "Dynamic Interaction of Eukaryotic Initiation Factor 4G1 (eIF4G1) with eIF4E and eIF1 Underlies Scanning-Dependent and -Independent Translation." Molecular and Cellular Biology 38, no. 18 (July 9, 2018). http://dx.doi.org/10.1128/mcb.00139-18.

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ABSTRACTTranslation initiation of most mRNAs involves m7G-cap binding, ribosomal scanning, and AUG selection. Initiation from an m7G-cap-proximal AUG can be bypassed resulting in leaky scanning, except for mRNAs bearing thetranslationinitiator ofshort 5′untranslated region (TISU) element. m7G-cap binding is mediated by the eukaryotic initiation factor 4E (eIF4E)-eIF4G1 complex. eIF4G1 also associates with eIF1, and both promote scanning and AUG selection. Understanding of the dynamics and significance of these interactions is lacking. We report that eIF4G1 exists in two complexes, either with eIF4E or with eIF1. Using an eIF1 mutant impaired in eIF4G1 binding, we demonstrate that eIF1-eIF4G1 interaction is important for leaky scanning and for avoiding m7G-cap-proximal initiation. Intriguingly, eIF4E-eIF4G1 antagonizes the scanning promoted by eIF1-eIF4G1 and is required for TISU. In mapping the eIF1-binding site on eIF4G1, we unexpectedly found that eIF4E also binds it indirectly. These findings uncover the RNA features underlying regulation by eIF4E-eIF4G1 and eIF1-eIF4G1 and suggest that 43S ribosome transition from the m7G-cap to scanning involves relocation of eIF4G1 from eIF4E to eIF1.

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Havkin-Solomon, Tal, Elad Itzhaki, Nir Joffe, Nina Reuven, Yosef Shaul, and Rivka Dikstein. "Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding." Nucleic Acids Research, April 18, 2023. http://dx.doi.org/10.1093/nar/gkad269.

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Abstract RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5’UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5’UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

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Ghosh, Sayan, Haitao Liu, Meysam Yazdankhah, Nadezda Stepicheva, Peng Shang, Tanuja Vaidya, Stacey Hose та ін. "βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity". Communications Biology 4, № 1 (24 лютого 2021). http://dx.doi.org/10.1038/s42003-021-01763-5.

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AbstractβA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as βA3 and βA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that βA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of βA3/A1-crystallin in metabolism of retinal astrocytes. We found that βA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but βA3-crystallin does not. Loss of βA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited βA1-knockdown (KD) mice, but not in βA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified βA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced βA1-crystallin and higher levels of PTP1B in the vitreous humor.

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Kim, Myung-Hwi, Boram Choi, Seok-Yeong Jang, Ji-Soo Choi, Sora Kim, Yubin Lee, Suejin Park, Sun-Jung Kwon, Jin-Ho Kang, and Jang-Kyun Seo. "The VP53 protein encoded by RNA2 of a fabavirus, broad bean wilt virus 2, is essential for viral systemic infection." Communications Biology 7, no. 1 (April 16, 2024). http://dx.doi.org/10.1038/s42003-024-06170-0.

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AbstractPlant viruses evolves diverse strategies to overcome the limitations of their genomic capacity and express multiple proteins, despite the constraints imposed by the host translation system. Broad bean wilt virus 2 (BBWV2) is a widespread viral pathogen, causing severe damage to economically important crops. It is hypothesized that BBWV2 RNA2 possesses two alternative in-frame translation initiation codons, resulting in the production of two largely overlapping proteins, VP53 and VP37. In this study, we aim to investigate the expression and function of VP53, an N-terminally 128-amino-acid-extended form of the viral movement protein VP37, during BBWV2 infection. By engineering various recombinant and mutant constructs of BBWV2 RNA2, here we demonstrate that VP53 is indeed expressed during BBWV2 infection. We also provide evidence of the translation of the two overlapping proteins through ribosomal leaky scanning. Furthermore, our study highlights the indispensability of VP53 for successful systemic infection of BBWV2, as its removal results in the loss of virus infectivity. These insights into the translation mechanism and functional role of VP53 during BBWV2 infection significantly contribute to our understanding of the infection mechanisms employed by fabaviruses.

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David, Maya, Tsviya Olender, Orel Mizrahi, Shira Weingarten-Gabbay, Gilgi Friedlander, Sara Meril, Nadav Goldberg, et al. "DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells." RNA, August 12, 2022, rna.079194.122. http://dx.doi.org/10.1261/rna.079194.122.

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Death Associated Protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate non-canonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knock-down (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing. A cohort of 68 genes showed decreased translation efficiency in DAP5 KD cells. Mass spectrometry confirmed decreased protein abundance of a significant portion of these targets. Among these was Kmt2d, a histone methylase previously shown to be essential for ESC differentiation and embryonic development. We found that nearly half of the cohort of DAP5 target mRNAs displaying reduced translation efficiency of their main coding sequences upon DAP5 KD contained upstream open reading frames (uORFs) that are actively translated independently of DAP5. This is consistent with previously suggested mechanisms by which DAP5 mediates leaky scanning through uORFs and/or re-initiation at the main coding sequence. Crosslinking protein-RNA immunoprecipitation experiments indicated that a significant subset of DAP5 mRNA targets bound DAP5, indicating that direct binding between DAP5 protein and its target mRNAs is a frequent but not absolute requirement for DAP5-dependent translation of the main coding sequence. Thus, we have extended DAP5’s function in translation of specific mRNAs in hESCs by a mechanism allowing translation of the main coding sequence following upstream translation of short ORFs.

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Yeckes, Alyson R., Aaron R. Victor, Zheng Zhu, Meena Narayanan, Bharani Srinivasan, Bethany Bruce, and Jonathan Kaye. "The Tox Gene Encodes Two Proteins with Distinct and Shared Roles in Gene Regulation." Journal of Immunology, April 28, 2023. http://dx.doi.org/10.4049/jimmunol.2200659.

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Abstract Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection–associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

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Bohlen, Jonathan, Mykola Roiuk, Marilena Neff, and Aurelio A. Teleman. "PRRC2 proteins impact translation initiation by promoting leaky scanning." Nucleic Acids Research, March 3, 2023. http://dx.doi.org/10.1093/nar/gkad135.

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Abstract Roughly half of animal mRNAs contain upstream open reading frames (uORFs). These uORFs can represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at the 5′ end and then scan for ORFs in a 5′-to-3′ fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important instance of post-transcriptional regulation that affects gene expression. Few molecular factors regulating or facilitating this process are known. Here we show that the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C impact translation initiation. We find that they bind eukaryotic translation initiation factors and preinitiation complexes, and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote leaky scanning past translation start codons, thereby promoting translation of mRNAs containing uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles.

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Shestakova, Ekaterina D., Victoria V. Smirnova, Ivan N. Shatsky, and Ilya M. Terenin. "Specific Mechanisms of Translation Initiation in Higher Eukaryotes: The eIF4G2 Story." RNA, December 14, 2022, rna.079462.122. http://dx.doi.org/10.1261/rna.079462.122.

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The eukaryotic initiation factor 4G2 (eIF4G2, DAP5, Nat1, p97) was discovered in 1997. Over the past two decades, dozens of papers have presented contradictory data on eIF4G2 function. Since its identification, eIF4G2 has been assumed to participate in noncanonical translation initiation mechanisms, but recent results indicate that it can be involved in scanning as well. In particular, eIF4G2 provides leaky scanning through some upstream open reading frames (uORFs), which are typical for long 5’ UTRs of mRNAs from higher eukaryotes. It is likely the protein can also help the ribosome overcome other impediments during scanning of the 5’ UTRs of animal mRNAs. This may explain the need for eIF4G2 in higher eukaryotes, as many mRNAs that encode regulatory proteins have rather long and highly structured 5’ UTRs. Additionally, they often bind to various proteins, which also hamper the movement of scanning ribosomes. This review discusses the suggested mechanisms of eIF4G2 action, denotes obscure or inconsistent results, and proposes ways to uncover other fundamental mechanisms in which this important protein factor may be involved in higher eukaryotes.

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