RNA structure profiling at single-cell resolution reveals new determinants of cell identity.

RNA structure is critical for multiple steps in gene regulation. However, how the structures of transcripts differ both within and between individual cells is unknown. Here we develop a SHAPE-inspired method called single-cell structure probing of RNA transcripts that enables simultaneous determination of transcript secondary structure and abundance at single-cell resolution. We apply single-cell structure probing of RNA transcripts to human embryonic stem cells and differentiating neurons. Remarkably, RNA structure is more homogeneous in human embryonic stem cells compared with neurons, with the greatest homogeneity found in coding regions. More extensive heterogeneity is found within 3' untranslated regions and is determined by specific RNA-binding proteins. Overall RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. We further discover a cell-type variable region of 18S ribosomal RNA that is associated with cell cycle and translation control. Our method opens the door to the systematic characterization of RNA structure-function relationships at single-cell resolution.

Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.

Genome-wide RNA structure changes during human neurogenesis modulate gene regulatory networks.

The distribution, dynamics, and function of RNA structures in human development are under-explored. Here, we systematically assayed RNA structural dynamics and their relationship with gene expression, translation, and decay during human neurogenesis. We observed that the human ESC transcriptome is globally more structurally accessible than differentiated cells and undergoes extensive RNA structure changes, particularly in the 3' UTR. Additionally, RNA structure changes during differentiation are associated with translation and decay. We observed that RBP and miRNA binding is associated with RNA structural changes during early neuronal differentiation, and splicing is associated during later neuronal differentiation. Furthermore, our analysis suggests that RBPs are major factors in structure remodeling and co-regulate additional RBPs and miRNAs through structure. We demonstrated an example of this by showing that PUM2-induced structure changes on LIN28A enable miR-30 binding. This study deepens our understanding of the widespread and complex role of RNA-based gene regulation during human development.

Determination of isoform-specific RNA structure with nanopore long reads.

Current methods for determining RNA structure with short-read sequencing cannot capture most differences between distinct transcript isoforms. Here we present RNA structure analysis using nanopore sequencing (PORE-cupine), which combines structure probing using chemical modifications with direct long-read RNA sequencing and machine learning to detect secondary structures in cellular RNAs. PORE-cupine also captures global structural features, such as RNA-binding-protein binding sites and reactivity differences at single-nucleotide variants. We show that shared sequences in different transcript isoforms of the same gene can fold into different structures, highlighting the importance of long-read sequencing for obtaining phase information. We also demonstrate that structural differences between transcript isoforms of the same gene lead to differences in translation efficiency. By revealing isoform-specific RNA structure, PORE-cupine will deepen understanding of the role of structures in controlling gene regulation.