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RIP-PEN-seq identifies a class of kink-turn RNAs as splicing regulators

Nature Biotechnology volume 42, pages 119–131 (2024)Cite this article

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Abstract

A kink-turn (K-turn) is a three-dimensional RNA structure that exists in all three primary phylogenetic domains. In this study, we 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.

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Fig. 1: A class of ncRNAs with a backward K-turn structure.
Fig. 2: Complex expression patterns of bktRNAs in tissues, cell lines and subcellular compartments and their evolution patterns.
Fig. 3: bktRNA1 guides the FBL protein to introduce 2′-O-methylation in U12 snRNA.
Fig. 4: Dysregulation of U12-type intron splicing in bktRNA1-depleted cells.
Fig. 5: Depletion of bktRNA1 affects U12 interactions with ZCRB1.
Fig. 6: Local regulation of intron splicing by bktRNAs.

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Data availability

All sequencing data that support the findings of this study have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus with the following accession numbers: GSE160970 for all HEK293T 15.5K RIP-PEN-seq; GSE182757 for all Hepa1-6 RIP-PEN-seq; GSE160636 for FBL and 15.5K CLASH-seq in HEK293T cells; GSE160887 for PEN-seq in HCT116, U-87 MG, Hela, HEK293T, HepG2 and K562 cells; GSE186849 for PEN-seq in 15.5K knockdown HEK293T cells; GSE182843 for PEN-seq in HEK293T and HCT116 cell fractions; GSE160515 for RNA-seq in bktRNA1 KO HCT116 cells; GSE182830 for RNA-seq in ZCRB1 knockdown HCT116 cells; GSE182759 for RNA-seq in 15.5K knockdown HEK293T cells; and GSE220470 for RIP-PEN-SHAPE-MaP in HEK293T cells. All data are available in the manuscript and in Supplementary Information and Source data files. There are no restriction on data availability. Source data are provided with this paper.

Code availability

The program kturnSeeker was written in the C++ programming language and is available from GitHub with no restrictions or conditions on access: https://github.com/sysu-software/kturnSeeker.

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Acknowledgements

We thank L. Huang from Sun Yat-sen Memorial Hospital for valuable suggestions on this manuscript. We thank Y. Zhang for sharing Sleeping Beauty transposon system. We thank all the staff from Sun Yat-sen University for their support and hard work during the COVID-19 pandemic. This work was supported, in part, by the National Key R&D Program of China (2019YFA0802202 (to J.Y.) and 2022YFA1303300 (to J.Y.)); the National Natural Science Foundation of China (32225011 (to J.Y.), 91940304 (to J.Y.), 31971228 (to J.Y.), 31770879 (to J.Y.), 31970604 (to L.Q.), 31900903 (to B.L.) and 32100467(to S.L.)); the Youth Science and Technology Innovation Talent of Guangdong TeZhi Plan (2019TQ05Y181 (to J.Y.)); funds from Guangzhou City (202002030351 (to J.Y.)); and Fundamental Research Funds for the Central Universities, Sun Yat-sen University (20lgpy112 (to B.L.) and 2021qntd26 (to B.L.)).

Author information

Author notes

  1. These authors contributed equally: Bin Li, Shurong Liu, Wujian Zheng, Anrui Liu, Peng Yu.

Authors and Affiliations

  1. MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Bin Li, Shurong Liu, Wujian Zheng, Anrui Liu, Peng Yu, Di Wu, Jie Zhou, Ping Zhang, Chang Liu, Qiao Lin, Jiayi Ye, Qiaojuan Huang, Hui Zhou, Lianghu Qu & Jianhua Yang

  2. The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China

    Wujian Zheng, Anrui Liu, Di Wu, Jie Zhou, Qiao Lin, Jiayi Ye, Simeng He & Jianhua Yang

  3. Department of Systems Biology, Beckman Research Institute of City of Hope, Monrovia, CA, USA

    Jianjun Chen

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Contributions

J.Y., B.L. and L.Q. conceived and designed the entire project. J.Y. and L.Q. designed and supervised the research. B.L., S.L., W.Z., A.L., P.Y., D.W., J.Z., P.Z., C.L., Q.L., J.Y., S.H., Q.H., H.Z. and J.Y. performed the experiments and/or data analyses. J.Y., B.L. and A.L. performed the genome-wide or transcriptome-wide data analyses. J.Y., B.L., S.L. and L.Q. contributed reagents/analytic tools and/or grant support. J.C. provided helpful discussions. J.Y., L.Q., B.L., S.L. and A.L. wrote and revised the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Lianghu Qu or Jianhua Yang.

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Competing interests

J.C. is a scientific advisory board member of Race Oncology. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Characterization of the RIP-PEN-seq technique.

a, Diagram of RNase H-based rRNA depletion for the construction of RIP-PEN-seq library. b, Western blotting analysis of the overexpression of 15.5K-FLAG protein in HEK293T cells. GAPDH serves as the loading control. c, RIP from HEK293T cells was performed using anti-FLAG and IgG. d, e, Meta-analyses of the RIP-PEN-seq results for the start (d) and end (e) sites of the forward K-turn RNAs (box C/D ncRNAs) in HEK293T cells. f, Genome-browser plot of RIP-PEN-seq (coverage, green; 5’-start, red; 3’-end, purple) for representative forward K-turn RNAs (box C/D snoRNAs) in the introns of GAS5. g, Computational workflow for analysis of the RIP-PEN-seq sequencing data and identification of candidate transcripts. h, KturnSeeker core algorithm workflow. KturnSeeker was developed to identify and quantify forward (fktRNAs) and backward ktRNAs (bktRNAs) from RIP-PEN-seq data. KturnSeeker can screen bktRNAs as well as fktRNAs by reverse searching the K-turn structure. i, Gene type distribution of forward ktRNAs identified from RIP-PEN-seq. CD-type represents C/D box-containing snoRNAs and scaRNAs. ACA-type represents snoRNAs or scaRNAs that only contain H/ACA boxes.

Source data

Extended Data Fig. 2 Structural characterization of bktRNAs.

a, The position and significance of motifs located within bktRNAs. The enriched motifs were identified by MEME software. The K-turn structural motif of bktRNAs is composed of two conserved sequence motifs: a CUGA motif often 4 nt downstream of the 5’ end and a UGAUG motif often 2 nt upstream of the 3’ end. The position p-value is defined as the probability that a random sequence would have a motif match score greater or equal to the sequence under test. b, Another twelve novel bktRNAs discovered from RIP-PEN-seq data. All novel bktRNAs had a CUGA motif that was often 4 nt downstream of the 5’ end and a UGAUG motif that was often 2 nt upstream of the 3’ end. These two sequence motifs were located within the K-turn structural motifs of bktRNAs. The 5’ motif (CUGA) and 3’ motif (UGAUG) are marked with black rectangles. The non-canonical A•G and G•A base pairs and the mismatch in the backward K-turn structure are also marked. c, Schematic overview of 15.5 K RIP-PEN-SHAPE-MaP. d, Secondary structure of consensus fktRNA (left panel, n = 98). Violin plots displaying the SHAPE reactivity across the forward K-turn structure (including 5’ C stem, internal (internal loop), 5’ NC stem, Loop, 3’ NC stem, and 3’ C stem), averaged across all known box C/D snoRNAs (right panel). The boxplots indicate the median and the upper and lower quartiles. e, The predicted secondary structure (upper panel) and SHAPE reactivity signal (lower panel) on fktRNA SNORD102 (also known as U102). The forward K-turn structure is indicated in the structure figure. The NC-stem and C-stem are marked with black and red underlines in the bar plot, respectively. The SHAPE reactivity signal was determined by RIP-PEN-SHAPE-MaP in this study.

Extended Data Fig. 3 The backward K-turn sequence composition, predicted functions and tissue-specific expression profile of bktRNAs.

a, The secondary structure of consensus forward K-turn RNA (fktRNA) and backward K-turn RNA (bktRNA). The nucleotide positions in the K-turn structure are named according to the nomenclature rules for the forward K-turn structure. b, Matrix plot showing the number of human bktRNAs with the indicated nucleotide in the 3b:3n sequences. c, Number of human bktRNAs with the four possible Watson-Crick base pairs in the -1b:-1n position. d, Matrix plot showing the number of mouse bktRNAs with the indicated nucleotide in the 3b:3n sequences. e, Number of mouse bktRNAs with the four possible Watson-Crick base pairs in the -1b:-1n position. f, Number of bktRNAs with or without m6A modification in humans and mice. g, Number of bktRNAs with or without m6A modification at the 1n position in humans and mice. h, Enrichment analysis of the bktRNA host protein-coding genes by Metascape software. i, Tissue-specific expression profiles of bktRNAs. The expression levels of bktRNAs are displayed in the rows and the tissues are shown in the columns. The rows and columns are sorted based on k-means clustering of bktRNA genes. The colour intensity represents the tissue-specific score (JS score) as calculated for each bktRNA using the csSpecificity function. Representative bktRNAs are indicated in the right panel.

Extended Data Fig. 4 Genomic characterization, expression, conservation, and secondary structure of bktRNA1.

a, Genome-browser plot of RIP-PEN-seq (coverage, blue; 5’-start, red; 3’-end, yellow) for bktRNA1, as well as the evolutionary conservation across 100 vertebrates (green). b, Secondary structure of bktRNA1 in the human genome was predicted by R-scape software. The SHAPE reactivities for each nucleotide were mapped to secondary structures using R2R software. The box H/ACA domain is indicated with a black dashed box, and the backward K-turn structure and the potential K-turn-like structure are marked with green dashed boxes. The NC stem and C stem are indicated with black lines. The blue boxes show the representative motifs. CAB, Cajal body box. c, The SHAPE reactivity signal on bktRNA1. The representative motifs are underlined in the bar plot. The SHAPE reactivity signal was determined by RIP-PEN-SHAPE-MaP in this study.

Extended Data Fig. 5 Secondary structures and subcellular localization of bktRNA1 and its interacting partner U12 snRNA.

a, Predicted conserved RNA structure of bktRNA1 determined by measuring pairwise covariations with R-scape software. The H/ACA domain is indicated with a black dashed box. The functional region paired with U12 snRNA is indicated with a blue dashed box. b, In situ co-localization of bktRNA1 with 15.5 K proteins and U12 snRNAs in HEK293T cells by fluorescent in situ hybridization (FISH) and immunofluorescence (IF) microscopy. White arrows indicate the signal detected by probes or antibody. c, In situ co-localization of bktRNA1 with 15.5 K proteins and U12 snRNAs in HCT116 cells by fluorescent in situ hybridization (FISH) and immunofluorescence (IF) microscopy. White arrows indicate the signal detected by probes or antibody.

Extended Data Fig. 6 Splicing efficiency analysis for wild-type and knockout bktRNA1.

a, Workflow for intron retention analysis in HCT116 and KO-bktRNA1 cells. b, Proportion of aberrantly retained U12- and U2-type introns (filtered by p < 0.05) in bktRNA1-deficient cells. c, Proportion of statistically significant changes (filtered by p < 0.05) in U12- and U2-type genes in bktRNA1-deficient cells. d, The ratio of spliced to unspliced pre-mRNA for U12-type introns was determined by qPCR in bktRNA1-deficient cells. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test. e, The ratio of spliced to unspliced pre-mRNA for U12-type and U2-type (GAPDH) introns was determined by qPCR in bktRNA1-rescued HCT116 KO-4 cells. f, The ratio of spliced to unspliced pre-mRNA for U12-type and U2-type (GAPDH) introns was determined by qPCR in SNORA12-rescued HCT116 KO-4 cells. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test. ns, no significance. g, The ratio of spliced to unspliced pre-mRNA for U12-type and U2-type (GAPDH) introns was determined by qPCR in artificial scaRNA-overexpressing HCT116 KO-4 cells. Data are presented as mean values +/- SEM (n = 3, biological replicates), two-tailed, paired t-test. ns, no significance.

Extended Data Fig. 7 Depletion of bktRNA1 affects the interaction between U12 and ZCRB1.

a, Western blots showing precipitation with each indicated antibody in wild-type (WT) and bktRNA1-deficient KO-4 (KO) cells. b, Native RIP was performed in wild-type (WT) and bktRNA1-deficient KO-4 (KO) cells using each indicated antibody or normal IgG antibody, after which qPCR was performed with primers recognizing minor splice snRNAs (U11, U12, U4atac, U5, U6atac). The percentage of RIP-enriched snRNAs was calculated relative to the input RNA. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test. ns, no significance. c, ZCRB1 RIP-enriched snRNAs were detected by Northern blotting in wild-type (WT) and bktRNA1-deficient KO-4 (KO) cells. U6 snRNA served as a negative control. d, ZCRB1 RIP-enriched snRNAs were detected by Northern blotting in bktRNA1-deficient and bktRNA1-rescued cells. U6 snRNA served as a negative control.

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Extended Data Fig. 8 ZCRB1 knockdown affects U12-type intron splicing.

a, qPCR (upper panel) and western blotting analysis (lower panel) of Dox-inducible ZCRB1 knockdown in HCT116 cells. GAPDH was used as an internal reference gene for qPCR, and GAPDH served as the loading control for western blotting. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test. b, c, Dot plots displaying the intron retention levels in a representative pairwise analysis of ZCRB1 knockdown and negative control cells. The red dots represent U12-type introns, and the blue dots represent U2-type introns. d, Proportion of aberrantly retained U2- and U12- type introns in ZCRB1 knockdown cells. The red boxes represent retained introns, and the blue boxes represent unretained introns. e, f, Cumulative fraction of the inclusion level difference between U12-type and U2-type introns in ZCRB1 knockdown and negative control cells. The P value on the cumulative plots of inclusion level differences were calculated using a two-sided Mann-Whitney-Wilcoxon test. g, Venn diagram showing the numbers of overlapping retained introns across four bktRNA1-deficient HCT116 cell lines and ZCRB1 knockdown cells. h, CCK-8 assay of HCT116 cells with bktRNA1 knockout. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test. i, Colony formation assay of HCT116 cells with bktRNA1 knockout. j, Quantitative analysis of colony formation assay in the indicated lines. Data are presented as mean values +/- SEM (n = 3, biological replicates), two-tailed, paired t-test. k, CCK-8 assay of HCT116 cells with ZCRB1 knockdown. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test. l, Colony formation assay of HCT116 cells with ZCRB1 knockdown. m, Quantitative analysis of colony formation assay in the indicated lines. Data are presented as mean values +/− SEM (n = 3, biological replicates), two-tailed, paired t-test.

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Unprocessed western blots and northern blots.

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Li, B., Liu, S., Zheng, W. et al. RIP-PEN-seq identifies a class of kink-turn RNAs as splicing regulators. Nat Biotechnol 42, 119–131 (2024). https://doi.org/10.1038/s41587-023-01749-0

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