RIP-PEN-seq – RNA immunoprecipitation coupled with sequencing of paired ends of ncRNAs

A kink-turn (K-turn) is a three-dimensional RNA structure that exists in all three primary phylogenetic domains. Researchers at Sun Yat-sen University have developed the RIP-PEN-seq method to identify the full-length sequences of RNAs bound by the K-turn binding protein 15.5K and discovered a previously uncharacterized class of RNAs with backward K-turn motifs (bktRNAs) in humans and mice. All bktRNAs share two consensus sequence motifs at their fixed terminal position and have complex folding properties, expression and evolution patterns. We found that a highly conserved bktRNA1 guides the methyltransferase fibrillarin to install RNA methylation of U12 small nuclear RNA in humans. Depletion of bktRNA1 causes global splicing dysregulation of U12-type introns by impairing the recruitment of ZCRB1 to the minor spliceosome. Most bktRNAs regulate the splicing of local introns by interacting with the 15.5K protein. Taken together, our findings characterize a class of small RNAs and uncover another layer of gene expression regulation that involves crosstalk among bktRNAs, RNA splicing and RNA methylation.

A class of ncRNAs with a backward K-turn structure

Fig. 1

a, Secondary structure of the consensus fktRNA and bktRNA. b, Procedure for the construction of RIP-PEN-seq libraries. c,d, Histogram showing the numbers of bktRNAs with different distances between the backward K-turn structure and the bktRNA 5′ end (c) or 3′ end (d). e, Enriched motifs identified in bktRNAs by MEME software. f, Gene model of bktRNA identified from RIP-PEN-seq data. g, Violin plots displaying the SHAPE reactivity across the backward K-turn structure (including 5′ NC stem, 5′ C stem, Loop, 3′ C stem, internal (internal loop) and 3′ NC stem), averaged across all bktRNAs (n = 22) in the RIP-PEN-SHAPE-MaP data. h,i, The predicted secondary structure of the backward K-turn (left panel) and SHAPE reactivity signal (right panel) on bktRNA1 (h) and bktRNA2 (i). The 5′ and 3′ motifs are indicated with black boxes in the structure figures. The NC-stem and C-stem are marked with black and red underlines in the bar plot, respectively. The SHAPE reactivity is calculated from merged n = 4 biological replicates. RT, reverse transcription.

Availability – The program kturnSeeker was written in the C++ programming language and is available from GitHub with no restrictions or conditions on access:

Li B, Liu S, Zheng W, Liu A, Yu P, Wu D, Zhou J, Zhang P, Liu C, Lin Q, Ye J, He S, Huang Q, Zhou H, Chen J, Qu L, Yang J. (2023) RIP-PEN-seq identifies a class of kink-turn RNAs as splicing regulators. Nat Biotechnol [Epub ahead of print]. [article]

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