Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs

Sachs, Matthew S.; Wang, Zhong; Gaba, Anthony; Peng, Fang; Belk, Jonathan Philip; Ganesan, Robin; Amrani, Nadia; Jacobson, Allan

doi:10.1016/s1046-2023(02)00013-0

Cited by 66 publications

(57 citation statements)

References 30 publications

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“…4, A (lane 4) and B). Observation of several bands in toeprint analysis (ϳϩ16 to ϩ18) has been reported by others (26,48) and is seen in cell extract (48).…”

Section: S Ribosomal Subunits Bind First To the Bydv Mrna 3ј-utr-elsupporting

confidence: 69%

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Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation

Sharma

Kraft²,

Miller

et al. 2015

Journal of Biological Chemistry

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Background: Barley yellow dwarf virus, a positive strand RNA virus, contains a 3Ј-translational enhancer element (BTE). Results: Ribosomes bind the 5Ј-UTR only in the presence of the 3Ј-element. Conclusion:The BTE is the site of initial ribosome recruitment. Significance: Ribosome recruitment to the 3Ј-BTE provides a feedback loop for the switch between translation and transcription of the viral mRNA.

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“…4, A (lane 4) and B). Observation of several bands in toeprint analysis (ϳϩ16 to ϩ18) has been reported by others (26,48) and is seen in cell extract (48).…”

Section: S Ribosomal Subunits Bind First To the Bydv Mrna 3ј-utr-elsupporting

confidence: 69%

“…The assay protocol for toeprinting in WGE was adapted from Ref. 48. Wheat germ extract reactions were prepared according to the Promega wheat germ in vitro translation kit (L-4380) as they were for use in translation assays, except that a complete amino acid mixture was used, and no [ 35 S]Met was used.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation

Sharma

Kraft²,

Miller

et al. 2015

Journal of Biological Chemistry

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“…Yeast were centrifuged and resuspended in YPG or glucose-containing medium, and a 2-L culture was grown for 6 h to a final OD of 1.5. Saccharomyces cerevisiae cell-free translation extracts were prepared using a modified previously described procedure (50). Small molecules were removed from S30 lysates using a Zeba Desalt Spin Column (Pierce) preequilibrated with Buffer A with 8.5% manniotol and 0.5 mM PMSF (51).…”

Section: Methodsmentioning

confidence: 99%

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

Lee

Burdeinick-Kerr

Whelan

2012

Proc. Natl. Acad. Sci. U.S.A.

148

142

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Initiation is the primary target of translational control for all organisms. Regulation of eukaryotic translation is traditionally thought to occur through initiation factors and RNA structures. Here, we characterize a transcript-specific translation initiation mechanism that is mediated by the ribosome. By studying vesicular stomatitis virus (VSV), we identify the large ribosomal subunit protein rpL40 as requisite for VSV cap-dependent translation but not bulk cellular or internal ribosome entry site-driven translation. This requirement is conserved among members of the order Mononegavirales, including measles virus and rabies virus. Polysome analyses and in vitro reconstitution of initiation demonstrate that rpL40 is required for 80S formation on VSV mRNAs through a cis-regulatory element. Using deep sequencing, we further uncover a subset of cellular transcripts that are selectively sensitive to rpL40 depletion, suggesting VSV may have usurped an endogenous translation pathway. Together, these findings demonstrate that the ribosome acts as a translational regulator outside of its catalytic role during protein synthesis.ribosome code | rhabdovirus | alternative translation | uba52 | paramyxovirus T ranslation initiation in eukaryotes proceeds generally by a cap-dependent scanning mechanism. The rate-limiting step in this process is recognition of the 5′ m 7 GpppN mRNA cap structure by eukaryotic translation initiation factor 4E (eIF4E) (1). The cap-binding complex recruits the small 40S subunit in complex with initiation factors, which then scans along the mRNA until it reaches the initiation codon AUG. Start codon recognition causes the release of the initiation factors, and the large 60S subunit joins, forming an elongation competent 80S complex (2, 3).The study of viral gene expression has uncovered several exceptions to this general mechanism of translation initiation. Poliovirus mRNA contains an ∼750-nt highly structured 5′ UTR that directly recruits the ribosome to an internal RNA site independently of the cap-recognition complex (4). Such internal ribosome entry sites (IRESs) are present in many RNA viruses, and those used by members of the Dicistroviridae family, including Plautia stali intestine virus and cricket paralysis virus, remarkably do not require any initiation factors or the initiating methionine tRNA (5-7). Investigation of the mechanism by which capped but nonpolyadenylated viral mRNAs are translated has also demonstrated that structures or viral proteins can negate the need for poly(A) binding protein during mRNA translation (8, 9). Such studies on translation initiation in viruses have invariably led to subsequent identification of cellular RNAs that are translated by similar mechanisms (10, 11).Replication of negative-strand RNA viruses in the order Mononegavirales induces profound host shutoff, and inhibition of cellular translation effectively suppresses the host immune response and antiviral immunity (12). However, the mechanistic basis of selective viral translation by these viruses...

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“…8A. Due to the size of the translation initiation apparatus, the position of the P site of the ribosome (the peptidyl-tRNA binding site that binds the tRNA carrying the growing peptide chain) is typically 15-17 nt upstream of the toeprint peak (69). This means that the fragment size given out by the capillary gel electrophoresis is 15-17 nt shorter than the actual size.…”

Section: L65mentioning

confidence: 99%

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

Tsotakos

Silveyra

Lin

et al. 2015

American Journal of Physiology-Lung Cellular and Molecular Phys

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Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

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Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs

Cited by 66 publications

References 30 publications

Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation

Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

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