Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide's structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The protocol permits the quantitative probing of thousands of RNAs at once, by several chemical modification reagents, on the time scale of a day using a table-top Illumina machine. This method and a software package MAPseeker (http://simtk.org/home/map_seeker) address several potential sources of bias, by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters, and avoiding problematic heuristics in prior algorithms. We hope that the step-by-step description of MAP-seq 1.0 will help other RNA mapping laboratories to transition from electrophoretic to next-generation sequencing methods and to further reduce the turnaround time and any remaining biases of the protocol.Comment: 22 pages, 5 figure

Fungal Pre-mRNA 3′-End Processing

3' end processing of messenger RNAs (mRNAs) is not only an essential step in eukaryotic gene expression, but it also impacts many other aspects of mRNA maturation and decay. A large portion of eukaryotic genes produce multiple mRNAs with different 3' ends through alternative cleavage/polyadenylation (APA). mRNA 3' processing and especially APA has been increasingly recognized as an important mechanism for gene regulation. Much of what we currently know about eukaryotic mRNA 3' processing came from studies using the genetically tractable yeast systems. Here we review the fungal mRNA 3' processing system by describing both the evolutionarily conserved mechanisms as well as the fungus-specific features