RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.
ABSTRACT We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.
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ABSTRACT: An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.Molecular Systems Biology 08/2014; 10(8). · 14.10 Impact Factor
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ABSTRACT: Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA(Arg(ICG)) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA(Arg(ICG)). Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA(Arg(ICG)) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA(Arg(ICG)). Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.RNA 07/2013; · 4.62 Impact Factor
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ABSTRACT: Memory formation requires learning based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP)-9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and thus is associated with learning processes in the mammalian brain. As the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed at elucidating regulation of MMP-9 gene expression in the mouse brain after fear learning. We show herein that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e., the amygdala, hippocampus and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, -42/-50 and -478/-486 bp AP-1 binding motifs of mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning.Journal of Biological Chemistry 05/2013; · 4.60 Impact Factor