Probing enzyme-dependent pseudouridylation using direct RNA sequencing to assess neuronal epitranscriptome plasticity.

Chemical modifications in mRNAs such as pseudouridine (psi) can regulate gene expression, although our understanding of the functional impact of individual psi modifications, especially in neuronal cells, is limited. We apply nanopore direct RNA sequencing to investigate psi dynamics under cellular perturbations in SH-SY5Y cells. We assign sites to psi synthases using siRNA-based knockdown. A steady-state enzyme-substrate model reveals a strong correlation between psi synthase and mRNA substrate levels and psi modification frequencies. Next, we performed either differentiation or lead-exposure to SH-SY5Y cells and found that, upon lead exposure, not differentiation, the modification frequency is less dependent on enzyme levels suggesting translational control. Finally, we compared the plasticity of psi sites across cellular states and found that plastic sites can be condition-dependent or condition-independent; several of these sites fall within transcripts encoding proteins involved in neuronal processes. Our psi analysis and validation enable investigations into the dynamics and plasticity of RNA modifications.

Enzymatic synthesis of RNA standards for mapping and quantifying RNA modifications in sequencing analysis.

Synthesis of RNA standards that contain an internal site-specific modification is important for mapping and quantification of the modified nucleotide in sequencing analysis. While RNA containing a site-specific modification can be readily synthesized by solid-state coupling for less than 100-mer nucleotides, longer RNA must be synthesized by enzymatic ligation in the presence of a DNA splint. However, long RNAs have structural heterogeneity, and those generated by in vitro transcription have 3'-end sequence heterogeneity, which together substantially reduce the yield of ligation. Here we describe a method of 3-part splint ligation that joins an in vitro transcribed left-arm RNA, an in vitro transcribed right-arm RNA, and a chemically synthesized modification-containing middle RNA, with an efficiency higher than previously reported. We report that the improved efficiency is largely attributed to the inclusion of a pair of DNA disruptors proximal to the ligation sites, and to a lesser extent to the homogeneous processing of the 3'-end of the left-arm RNA. The yields of the ligated long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.

Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing.

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.

Unidirectional single-file transport of full-length proteins through a nanopore.

The electrical current blockade of a peptide or protein threading through a nanopore can be used as a fingerprint of the molecule in biosensor applications. However, threading of full-length proteins has only been achieved using enzymatic unfolding and translocation. Here we describe an enzyme-free approach for unidirectional, slow transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore, α-hemolysin (narrowest part is ~1.4 nm in diameter), and a high concentration guanidinium chloride buffer enables unidirectional, single-file protein transport propelled by an electroosmotic effect. We show that the mean protein translocation velocity depends linearly on the applied voltage and translocation times depend linearly on length, resembling the translocation dynamics of ssDNA. Using a supervised machine-learning classifier, we demonstrate that single-translocation events contain sufficient information to distinguish their threading orientation and identity with accuracies larger than 90%. Capture rates of protein are increased substantially when either a genetically encoded charged peptide tail or a DNA tag is added to a protein.

Discrimination of RNA fiber structures using solid-state nanopores.

RNA fibers are a class of biomaterials that can be assembled using HIV-like kissing loop interactions. Because of the programmability of molecular design and low immunorecognition, these structures present an interesting opportunity to solve problems in nanobiotechnology and synthetic biology. However, the experimental tools to fully characterize and discriminate among different fiber structures in solution are limited. Herein, we utilize solid-state nanopore experiments and Brownian dynamics simulations to characterize and distinguish several RNA fiber structures that differ in their degrees of branching. We found that, regardless of the electrolyte type and concentration, fiber structures that have more branches produce longer and deeper ionic current blockades in comparison to the unbranched fibers. Experiments carried out at temperatures ranging from 20-60 °C revealed almost identical distributions of current blockade amplitudes, suggesting that the kissing loop interactions in fibers are resistant to heating within this range.

Transcriptional repression by FEZF2 restricts alternative identities of cortical projection neurons.

Projection neuron subtype identities in the cerebral cortex are established by expressing pan-cortical and subtype-specific effector genes that execute terminal differentiation programs bestowing neurons with a glutamatergic neuron phenotype and subtype-specific morphology, physiology, and axonal projections. Whether pan-cortical glutamatergic and subtype-specific characteristics are regulated by the same genes or controlled by distinct programs remains largely unknown. Here, we show that FEZF2 functions as a transcriptional repressor, and it regulates subtype-specific identities of both corticothalamic and subcerebral neurons by selectively repressing expression of genes inappropriate for each neuronal subtype. We report that TLE4, specifically expressed in layer 6 corticothalamic neurons, is recruited by FEZF2 to inhibit layer 5 subcerebral neuronal genes. Together with previous studies, our results indicate that a cortical glutamatergic identity is specified by multiple parallel pathways active in progenitor cells, whereas projection neuron subtype-specific identity is achieved through selectively repressing genes associated with alternate identities in differentiating neurons.