A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

Regulation of Co-transcriptional Pre-mRNA Splicing by m6A through the Low-Complexity Protein hnRNPG.

N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.

Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis.

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.