Massive functional mapping of a 5′-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing

Nucleic Acids Research, Jul 2013

We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5′-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure–function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.

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Massive functional mapping of a 5′-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing

Erik Holmqvist 0 Johan Reimega rd 0 E. Gerhart H. Wagner 0 0 Department of Cell and Molecular Biology, Biomedical Center, Uppsala University , SciLifeLab Uppsala, Box 596, S-75124 Uppsala, Sweden We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and highthroughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 50UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescenceactivated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins. - INTRODUCTION Forward and reverse genetics methods are valuable tools to link phenotypes to DNA sequences. Forward genetics identifies nucleotide changes that cause a phenotypic change, and reverse genetics identifies the phenotype associated with a particular mutation. In reverse genetics, site-directed mutagenesis can be used to, for example, pinpoint nucleotides in DNA/RNA sequences at which regulators bind and can assess RNA structure function relationships [e.g. (1,2)]. Random mutagenesis by polymerase chain reaction (PCR) under error-prone conditions is a powerful method for creating large pools of mutants (3). For instance, error-prone PCR followed by fluorescence-activated cell sorting (FACS) analysis has been used to generate variants of the green fluorescent protein (GFP) with increased intensity and more efficient folding (4). Related to the work presented here, errorprone PCR followed by phenotypic screening has identified base changes that affect expression and stability of the small RNA (sRNA) MicA, as well as MicA-dependent post-transcriptional regulation (5). Even though such approaches have turned out to be successful in identifying functionally important nucleotides, they are tedious and suffer from low throughput, as each mutant needs to be phenotypically assayed one by one. To increase throughput in reverse genetics, several recent articles have described methods for scoring effects on gene expression from large numbers of sequence variants. The RNA-ID method was designed to study cis-regulatory RNA sequences in yeast; short random sequences were inserted into an messenger RNA (mRNA), and effects on translation efficiency were monitored by FACS of fluorescent protein expression (6). Kudla et al. (7) used a library of 154 synthetic variants of gfp to study gene expression changes arising from synonymous mutations. Another article reported on a multiple mutation-, FACS- and high-throughput-sequencing method used for mapping protein binding and its energetics in transcriptional regulation (8). Two additional publications also describe similar methods for transcriptional regulation (9,10) and use mRNA abundance measurements as readout for gene expression. However, changes in DNA sequences that involve insertions (6) may introduce unwanted effects arising from different sequence lengths of the analyzed variants. Additionally, mRNA abundance does not always accurately report on a mutations effect on gene expression, as mRNA and protein levels are not always correlated, and protein expression often is predominantly regulated at the post-transcriptional level (9,11). For instance, studies of several bacterial mRNAs showed that sRNA-mediated regulation can give altered protein levels without significantly affecting mRNA levels (12,13). For functional screening and mapping of high numbers of mutations in single experiments, we present here a method that combines saturation mutagenesis, phenotypic cell sorting and high-throughput sequencing. This method is particularly powerful for studies of post-transcriptional regulation, but it is easily adaptable for studies of transcriptional regulation as well. Our method does not rely on insertion of sequences but generates nucleotide substitutions, eliminating the risk of unwanted effects throu (...truncated)


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Erik Holmqvist, Johan Reimegård, E. Gerhart H. Wagner. Massive functional mapping of a 5′-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing, Nucleic Acids Research, 2013, pp. e122-e122, 41/12, DOI: 10.1093/nar/gkt267