CaltechAUTHORS
  A Caltech Library Service

PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications

Shi, Junchao and Zhang, Yunfang and Tan, Dongmei and Zhang, Xudong and Yan, Menghong and Zhang, Ying and Franklin, Reuben and Shahbazi, Marta and Mackinlay, Kirsty and Liu, Shichao and Kuhle, Bernhard and James, Emma R. and Zhang, Liwen and Qu, Yongcun and Zhai, Qiwei and Zhao, Wenxin and Zhao, Linlin and Zhou, Changcheng and Gu, Weifeng and Murn, Jernej and Guo, Jingtao and Carrell, Douglas T. and Wang, Yinsheng and Chen, Xuemei and Cairns, Bradley R. and Yang, Xiang-lei and Schimmel, Paul and Zernicka-Goetz, Magdalena and Cheloufi, Sihem and Zhang, Ying and Zhou, Tong and Chen, Qi (2021) PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nature Cell Biology, 23 (4). pp. 424-436. ISSN 1465-7392. doi:10.1038/s41556-021-00652-7. https://resolver.caltech.edu/CaltechAUTHORS:20210211-165540246

[img] PDF (Supplementary Figs. 1–3) - Supplemental Material
See Usage Policy.

23MB
[img] PDF (Reporting Summary) - Supplemental Material
See Usage Policy.

1MB
[img] MS Excel (Supplementary Tables) - Supplemental Material
See Usage Policy.

22MB
[img] MS Excel (Source Data Fig. 1) - Supplemental Material
See Usage Policy.

12kB
[img] Image (JPEG) (Source Data Fig. 1) - Supplemental Material
See Usage Policy.

677kB
[img] MS Excel (Source Data Fig. 2) - Supplemental Material
See Usage Policy.

14kB
[img] Image (JPEG) (Source Data Fig. 2) - Supplemental Material
See Usage Policy.

591kB
[img] MS Excel (Source Data Fig. 3) - Supplemental Material
See Usage Policy.

16kB
[img] MS Excel (Source Data Fig. 5) - Supplemental Material
See Usage Policy.

910kB
[img] Image (JPEG) (Source Data Fig. 5) - Supplemental Material
See Usage Policy.

833kB
[img] MS Excel (Source Data Fig. 6) - Supplemental Material
See Usage Policy.

34kB
[img] MS Excel (Source Data Extended Data Fig. 1) - Supplemental Material
See Usage Policy.

14kB
[img] Image (JPEG) (Source Data Extended Data Fig. 2) - Supplemental Material
See Usage Policy.

240kB
[img] MS Excel (Source Data Extended Data Fig. 5) - Supplemental Material
See Usage Policy.

65kB
[img] Image (JPEG) (Source Data Extended Data Fig. 9) - Supplemental Material
See Usage Policy.

1MB
[img] Image (JPEG) (Extended Data Fig. 1: Reads summary and length distributions of different sncRNA category under Traditional RNA-seq, AlkB-facilitated RNA-seq, T4PNK-facilitated RNA-seq, and PANDORA-seq) - Supplemental Material
See Usage Policy.

426kB
[img] Image (JPEG) (Extended Data Fig. 2: Evaluation of Northern blot probe efficiency on synthesized targets (that is, rsRNA-28S-1, 5’tsRNAGlu, let-7i, mir-122, mir-21) - Supplemental Material
See Usage Policy.

80kB
[img] Image (JPEG) (Extended Data Fig. 3: Annotation of mouse piRNA in non-germ cell tissue/cell types is not stable when 1–3 mismatches are allowed) - Supplemental Material
See Usage Policy.

477kB
[img] Image (JPEG) (Extended Data Fig. 4: Scattered plot comparison of profile changes in tsRNAs and rsRNAs compared to miRNAs under different treatment protocol) - Supplemental Material
See Usage Policy.

509kB
[img] Image (JPEG) (Extended Data Fig. 5: The tsRNA responses to AlkB, T4PNK and PANDORA-seq in regard to different tsRNA origin (5’tsRNA, 3’tsRNA, 3’tsRNA with CCA end, and internal tsRNAs) - Supplemental Material
See Usage Policy.

284kB
[img] Image (JPEG) (Extended Data Fig. 6: Overall length mapping of tsRNA reads in genomic and mitochondrial tRNA under different RNA-seq protocol) - Supplemental Material
See Usage Policy.

382kB
[img] Image (JPEG) (Extended Data Fig. 7: The miRNAs that showing sensitive response to PANDORA-seq are in fact rsRNAs) - Supplemental Material
See Usage Policy.

383kB
[img] Image (JPEG) (Extended Data Fig. 8: The pairwise comparison matrices showing the differential expression pattern of rsRNAs under different RNA-seq protocol across tissues and cells) - Supplemental Material
See Usage Policy.

468kB
[img] Image (JPEG) (Extended Data Fig. 9: Northern blot analyses of tsRNA/rsRNA (that is, tsRNAAla, tsRNAArg, tsRNAGlu, tsRNAHis, tsRNALys and rsRNA-28S-1) changes during mESC to EB differentiation) - Supplemental Material
See Usage Policy.

176kB
[img] Image (JPEG) (Extended Data Fig. 10: Expression heatmap of the differentially expressed genes from representative GOBP terms in Day6 and Enriched GOBP terms of differential expressed genes in Day3 EBs after tsRNA/rsRNA transfection) - Supplemental Material
See Usage Policy.

590kB

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20210211-165540246

Abstract

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs—mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)—that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/s41556-021-00652-7DOIArticle
https://rdcu.be/ciLcOPublisherFree ReadCube access
https://doi.org/10.1038/s41556-021-00687-wDOIErratum
https://rdcu.be/cj1MGPublisherFree ReadCube access - Erratum
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144666Related ItemData
https://figshare.com/articles/dataset/_/14033003Related ItemData
https://github.com/junchaoshi/sports1.1Related ItemCode
ORCID:
AuthorORCID
Shi, Junchao0000-0002-1548-5069
Shahbazi, Marta0000-0002-1599-5747
Kuhle, Bernhard0000-0002-0501-1150
Zhai, Qiwei0000-0002-9617-7403
Zhao, Linlin0000-0002-8821-4198
Zhou, Changcheng0000-0001-7649-0710
Carrell, Douglas T.0000-0002-6471-2803
Wang, Yinsheng0000-0001-5565-283X
Chen, Xuemei0000-0002-5209-1157
Cairns, Bradley R.0000-0002-9864-8811
Yang, Xiang-lei0000-0003-2554-084X
Schimmel, Paul0000-0002-0448-022X
Zernicka-Goetz, Magdalena0000-0002-7004-2471
Cheloufi, Sihem0000-0002-1726-4796
Zhou, Tong0000-0003-2361-1931
Chen, Qi0000-0001-6353-9589
Alternate Title:PANDORA-seq: expanding the functional small RNA repertoire by overcoming RNA modifications
Additional Information:© 2021 Nature Publishing Group. Received 13 July 2020; Accepted 23 February 2021; Published 05 April 2021. We thank T. Lowe at the University of California, Santa Cruz for early discussion on the project, and Z. Li from the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences for assistance with operating the mass spectrometer. This work is in part supported by MOST (2019YFA0802600 to Ying Zhang (Chinese Academy of Sciences) and Yunfang Zhang; 2018YFC1004500 to Ying Zhang (Chinese Academy of Sciences) and M.Y.), startup funds from the University of California, Riverside (to Q.C. and S.C.) and the NIH (R01HD092431 to Q.C.; R01ES032024 to Q.C. and T.Z.; P50HD098593 to T.Z. and Q.C.; R35GM128854 to L. Zhao). This work includes data generated at the University of California, San Diego IGM Genomics Center funded by the NIH (P30DK063491, P30CA023100 and P30DK120515). Q.Z. is funded by the NSFC (31630037). Ying Zhang (University of California, Riverside) is funded by a State Scholarships Fund (201908500039). Yunfang Zhang is funded by the NSFC (82022029) and the Natural Science Foundation of Chongqing (cstc2019jcyjjqX0010). M.Y. is funded by the NSFC (31670830) and is a fellow of the Innovative Research Team of High-Level Local Universities in Shanghai. M.S. is funded by an Advanced EMBO fellowship. K.M. is funded by a BBSRC scholarship. Work in the laboratory of M.Z.-G. is funded by the Wellcome Trust (207415/Z/17/Z), ERC (669198) and Open Philanthropy. R.F. is supported by UC Riverside’s Eugene Cota-Robles Fellowship. Data availability: RNA-seq datasets have been deposited in the Gene Expression Omnibus under the accession code GSE144666. LC-MS/MS data have been deposited in Figshare (https://figshare.com/articles/dataset/_/14033003). All other data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper. Code availability: The sncRNA annotation pipeline SPORTS1.1 is available from GitHub (https://github.com/junchaoshi/sports1.1). The scripts used for data processing and statistical analysis were written in Perl or R and are available upon reasonable request. These authors contributed equally: Junchao Shi, Yunfang Zhang, Dongmei Tan, Xudong Zhang, Menghong Yan, Ying Zhang, Reuben Franklin. Author Contributions: Q.C., T.Z. and J.S. designed the project. Yunfang Zhang, D.T. and J.S. developed and optimized the enzymatic treatment protocol for PANDORA-seq. J.S., T.Z., Ying Zhang (Chinese Academy of Sciences) and Q.C. designed and developed the scope of data analyses. S.C., J.M. and R.F. generated iPSCs from MEFs and contributed to related analyses. X.Z. and R.F. performed the functional assays of mESCs under the supervision of S.C. and Q.C. J.S. and T.Z. developed the computational tools and analysed all of the datasets with input from Ying Zhang (Chinese Academy of Sciences) and Q.C. X.Z. and Ying Zhang (University of California, Riverside) developed and performed northern blot analyses for tissues/cells with help from D.T. and S.L. Yunfang Zhang tested and validated T4PNK’s effect in improving adapter ligation. M.Y. and X.Z. contributed to the LC-MS/MS RNA modification analyses with the help from Y.W. M.Y. designed and generated the AlkB plasmid and generated the AlkB enzyme with help from W.Z., Q.Z. and L. Zhao. L. Zhang and Y.Q. collected mature sperm samples under the supervision of Ying Zhang (Chinese Academy of Sciences). M.S., K.M. and M.Z.-G. performed experiments to contribute mESCs, primed hESCs and naive hESCs for analyses. B.R.C. contributed to data interpretation in regard to piRNAs and rsRNAs and the Discussion section, with input from D.T.C., J.G. and E.R.J. X.C. contributed to data interpretation in regard to miRNA and miRBase. P.S., X.-l.Y. and B.K. contributed to data interpretation of mitochondrial tsRNAs and discussed the evolutionary aspects. L. Zhao, C.Z., W.G., D.T.C., J.G. and E.R.J. contributed to the interpretation and discussion of data. Q.C. T.Z., Ying Zhang (Chinese Academy of Sciences) and J.S. wrote the main manuscript and integrated input from all authors. The authors declare no competing interests. Peer review information: Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work.
Errata:Shi, J., Zhang, Y., Tan, D. et al. Author Correction: PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nat Cell Biol (2021). https://doi.org/10.1038/s41556-021-00687-w
Funders:
Funding AgencyGrant Number
Ministry of Science and Technology (Taipei)2019YFA0802600
Chinese Academy of SciencesUNSPECIFIED
Ministry of Science and Technology (Taipei)2018YFC1004500
University of California, RiversideUNSPECIFIED
NIHR01HD092431
NIHR01ES032024
NIHP50HD098593
NIHR35GM128854
NIHP30DK063491
NIHP30CA023100
NIHP30DK120515
National Natural Science Foundation of China31630037
State Scholarships Fund (China)201908500039
National Natural Science Foundation of China82022029
Natural Science Foundation of Chongqingcstc2019jcyjjqX0010
National Natural Science Foundation of China31670830
European Molecular Biology Organization (EMBO)UNSPECIFIED
Biotechnology and Biological Sciences Research Council (BBSRC)UNSPECIFIED
Wellcome Trust207415/Z/17/Z
European Research Council (ERC)669198
Open PhilanthropyUNSPECIFIED
Subject Keywords:Non-coding RNAs; Reprogramming; RNA sequencing; Small RNAs
Issue or Number:4
DOI:10.1038/s41556-021-00652-7
Record Number:CaltechAUTHORS:20210211-165540246
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210211-165540246
Official Citation:Shi, J., Zhang, Y., Tan, D. et al. PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nat Cell Biol 23, 424–436 (2021). https://doi.org/10.1038/s41556-021-00652-7
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108027
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:16 Apr 2021 18:32
Last Modified:05 May 2021 19:23

Repository Staff Only: item control page