Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand.

Nanopore sequencing is one of only a few methods that can potentially determine the amino acid sequence of individual protein molecules as these are passed through a pore sensor. However, mechanisms for unfolding and translocation of proteins are still unavailable to date. Here we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We combine a chemically resistant biological nanopore platform with a high concentration guanidinium chloride buffer to achieve unidirectional, single-file protein transport that is propelled by a giant electro-osmotic effect, as revealed by molecular dynamics simulations and confirmed experimentally. Remarkably, we observed that protein velocities are uniform regardless of the protein sequence, which allows the identification and discrimination among proteins based on their electrical signatures, as well as to distinguish protein signatures by their threading orientation (N-to-C vs. C-to-N terminus). With average transport velocities of 10 µs per amino acid, our method can enable direct, enzyme-free protein fingerprinting and protein sequencing when combined with a high-resolution pore and high-speed nanopore readout.

Highlights d FEZF2 specifies subtype-specific fates in newly born neurons d FEZF2 functions as a transcriptional repressor to specify cell fate d FEZF2 specifies cell fates by repressing alternate cell-typespecific genes d FEZF2 and TLE4 are co-repressors in corticothalamic projection neurons