Control of mammalian translation by mRNA structure near caps

Babendure, Jeremy R.; Babendure, Jennie L.; Ding, Jian-Hua; Tsien, Roger Y.

doi:10.1261/rna.2309906

Cited by 292 publications

(340 citation statements)

References 35 publications

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“…Further, it should be noted that we also observed a significant decrease in GLuc reporter activity of the 5 ′ UTR-HP reporter construct in comparison with the unmodified or ATG-HP GLuc construct, even in the absence of Csy4. This is likely due to decreased efficiency of translational initiation by incorporation of a RNA hairpin structure in the 5 ′ UTR region (Kozak 1986(Kozak , 1989Babendure et al 2006). These results are consistent in part with studies in S. cerevisiae, B. subtilis, and E. coli (Qi et al 2012).…”

Section: Resultssupporting

confidence: 86%

Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4

Borchardt

Huang

et al. 2015

RNA

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The bacterial CRISPR endoribonuclease Csy4 has recently been described as a potential RNA processing tool. Csy4 recognizes substrate RNA through a specific 28-nt hairpin sequence and cleaves at the 3 ′ end of the stem. To further explore applicability in mammalian cells, we introduced this hairpin at various locations in mRNAs derived from reporter transgenes and systematically evaluated the effects of Csy4-mediated processing on transgene expression. Placing the hairpin in the 5 ′ UTR or immediately after the start codon resulted in efficient degradation of target mRNA by Csy4 and knockdown of transgene expression by 20-to 40-fold. When the hairpin was incorporated in the 3 ′ UTR prior to the poly(A) signal, the mRNA was cleaved, but only a modest decrease in transgene expression (∼2.5-fold) was observed. In the absence of a poly(A) tail, Csy4 rescued the target mRNA substrate from degradation, resulting in protein expression, which suggests that the cleaved mRNA was successfully translated. In contrast, neither catalytically inactive (H29A) nor binding-deficient (R115A/R119A) Csy4 mutants were able to exert any of the effects described above. Generation of a similar 3 ′ end by RNase P-mediated cleavage was unable to rescue transgene expression independent of Csy4. These results support the idea that the selective generation of the Csy4/hairpin complex resulting from cleavage of target mRNA might serve as a functional poly(A)/poly(A) binding protein (PABP) surrogate, stabilizing the mRNA and supporting translation. Although the exact mechanism(s) remain to be determined, our studies expand the potential utility of CRISPR nucleases as tools for controlling mRNA stability and translation.

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Section: Resultssupporting

confidence: 86%

Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4

Borchardt

Huang

et al. 2015

RNA

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“…The 5Ј-untranslated region of MT-1E mRNA is unique among human MT isoforms in that it contains a stable 12-bp GC-rich stem-loop structure positioned 26 bases from its 5Ј cap. The stability of the stem loop and its location are consistent with reduced translation efficiency (82). No other MT mRNA has a similar predicted stemloop as stable as that of MT-1E.…”

Section: Discussionsupporting

confidence: 56%

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Mehus

Muhonen

Garrett

et al. 2014

Molecular & Cellular Proteomics

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Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14 N-or 15 N-iodoacetamide. Absolute quantitation was achieved using 15 Niodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue-and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptornegative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity. Molecular & Cellular

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“…Thus, none of the constructs affect the coding region of nsP4 or its stop codons, which are positioned at the beginning of the sgmRNA sequence. The hairpin hp has a predicted minimum free energy ΔG of −45.30 kcal/mol (RNAfold, University of Vienna) and should not be melted by preinitiation complexes (Kozak 1990(Kozak , 1991Babendure et al 2006). Thus, the placement of this hairpin before the initiation codon hampers translation in the event that scanning occurs (Kozak 1990).…”

Section: Mechanism Of Protein Synthesis Initiation Directed By Sinv Smentioning

confidence: 99%

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

García-Moreno

Sanz

Carrasco

2014

RNA

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Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.

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Control of mammalian translation by mRNA structure near caps

Cited by 292 publications

References 35 publications

Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4

Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA

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